Methods and compositions for the treatment of cancer

ABSTRACT

Described herein are methods and compositions relating to the treatment of cancer, e.g., breast cancer, using, e.g., aptamer-siRNA chimera molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of co-pendingU.S. application Ser. No. 15/506,010 filed Feb. 23, 2017, which is a 35U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/US15/047449 filed Aug. 28, 2015, which designatesthe U.S. and claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 62/043,803 filed Aug. 29, 2014, the contents of whichare incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberW81XWH-09-1-0058 awarded by the U.S. Department of the Army. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 28, 2015, isnamed 701039-082401-PCT_SL.txt and is 8,984 bytes in size.

TECHNICAL FIELD

The technology described herein relates to chimeric molecules comprisingan EpCAM binding-molecule and an inhibitory nucleic acid and methods ofusing such compositions for the treatment of cancer, e.g. epithelialcancer.

BACKGROUND

RNA interference (RNAi) has been explored for therapeutic use inreducing gene expression in the liver. However, the liver is unique inbeing easy to transfect with RNAi molecules. Delivery of small RNAs andresulting gene knockdown in other tissues continues to be inefficientand ultimately ineffective. In particular, the delivery roadblock is amajor obstacle to harnessing RNAi to treat cancer.

SUMMARY

As described herein, the inventors have developed novel chimericaptamer-siRNA molecules (AsiCs). These AsiC's target cancer cell markersto direct the siRNA specifically to the cancer cells, increasingdelivery efficacy and therapeutic effectiveness while reducing thepotential for side effects.

In one aspect, described herein is a chimeric molecule comprising acancer marker-binding aptamer domain and an inhibitory nucleic aciddomain. In some embodiments, the cancer marker is EpCAM or EphA2. Insome embodiments, the inhibitory nucleic acid specifically binds to agene product upregulated in a cancer cell. In some embodiments, theinhibitory nucleic acid inhibits the expression of a gene selected fromthe group consisting of: Plk1; MCL1; EphA2; PsmA2; MSI1; BMI1; XBP1;PRPF8; PFPF38A; RBM22; USP39; RAN; NUP205; and NDC80. In someembodiments, the cancer marker is EpCAM and the inhibitory nucleic aciddomain inhibits the expression of Plk1.

In some embodiments, the molecule is an aptamer-siRNA chimera (AsiC). Insome embodiments, the cancer marker-binding aptamer domain comprises thesequence of SEQ ID NO: 33. In some embodiments, the cancermarker-binding aptamer domain consists essentially of the sequence ofSEQ ID NO: 33. In some embodiments, the inhibitory nucleic acid domaincomprises the sequence of SEQ ID NO: 2. In some embodiments, theinhibitory nucleic acid domain consists essentially of the sequence ofSEQ ID NO: 2. In some embodiments, the molecule comprises the sequenceof one of SEQ ID NOs: 1-3. In some embodiments, the molecule consistsessentially of the sequence of one of SEQ ID NOs: 1-3.

In some embodiments, the 3′ end of the molecule comprises dTdT. In someembodiments, the molecule comprises at least one 2′-F pyrimidine.

In one aspect, described herein is a pharmaceutical compositioncomprising a chimeric molecule as described herein and apharmaceutically acceptable carrier. In some embodiments, thecomposition comprises at least two chimeric molecules as describedherein wherein the chimeric molecules have different aptamer domainsand/or inhibitory nucleic acid domains. In some embodiments, thedifferent aptamer or inhibitory nucleic acid domains recognize differenttargets. In some embodiments, the different aptamer or inhibitorynucleic acid domains have sequences and recognize the same target.

In one aspect, described herein is a method of treating cancer, themethod comprising administering a chimeric molecule and/or compositionas described herein. In some embodiments, the cancer is an epithelialcancer or breast cancer. In some embodiments, the breast cancer istriple-negative breast cancer. In some embodiments, the administrationis subcutaneous. In some embodiments, the subject is furtheradministered an additional cancer treatment. In some embodiments, thecancer treatment is paclitaxel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H demonstrate that EpCAM aptamer specifically targets Basal Abreast cancer cells. Design of EpCAM-AsiC, containing an EpCAM aptamerand a PLK1 siRNA (sense strand disclosed as SEQ ID NO: 1 and antisensestrand disclosed as SEQ ID NO: 2) (FIG. 1C). Epithelial breast cancercell line (BPLER) over express EpCAM protein compared to normal breastepithelial cell line (BPE) (FIGS. 1A-1B). EpCAM-AsiC targeting GFP wasAlexa647 or Cy3 labeled at the 3′ end of the antisense siRNA strand andincubated with BPLER and BPE cells. Uptake was assessed 24 hours laterby flow cytometry (FIG. 1D). Data are representative of 3 independentexperiments. Cy3 and Alexa647-labeled EpCAM-AsiC was taken up by MB468and BPLER (EpCAM+ cells) respectively and not by BPE (EpCAM−). MFI ofeach peak is shown. To test for gene silencing, BPLER and BPE weretreated with EpCAM-AsiC targeting GFP (4 μM) and compared toTransfection controls using Dharmafect and GFP-siRNA (100 nM). Knockdownwas assessed by flow cytometry 72 hours after incubation. Controls weremock and Dhrmafect only treatment (lipid). (n=4) (FIG. 1D). EpCAM-AsiCtargeting AKT1 selectively knocks-down AKT1 mRNA (FIG. 1E) and protein(FIG. 1F) expression in basal A and luminal breast cancer cell lines andnot in basal B or human fibroblasts (hFb). Transfection with siRNAtargeting AKT1 induces gene knockdown in all cell lines, while treatmentwith EpCAM-AsiC targeting GFP doesn't effect AKT1 mRNA and proteinlevels (* p<0.05, p<0.01). Plots of AKT1 Protein and gene Knockdowncomparing the effect of EpCAM-AsiC to siRNA transfection. EpCAM-AsiCinduced knockdown correlates with EpCAM expression (FIG. 1E-H). (n=3;mean±SEM normalized to mock; *P<0.05, **P<0.01, 2-tailed t test).

FIGS. 2A-2E demonstrate that EpCAM AsiC targeting PLK1 specificallyinhibits cell proliferation in Basal A breast cancer cells. The effectof EpCAM-AsiC targeting PLK1 on cell proliferation was tested on 10breast cancer cell lines representative of basal A, B and luminal celllines using cell-titer-glo assay (CTG). EpCAM-AsiC targeting PLK1decreased cell proliferation in both basal A and luminal cell lineswhile having no effect on basal B cells (FIGS. 2A, 2C). A correlationwas seen between EpCAM expression levels and cell viability (FIG. 2B).Basal A (EpCAM+GFP−) cell were co-cultured with BPE (EpCAM-GFP+) cellsand treated with EpCAM-AsiC targeting PLK1 or untreated. Untreatedco-culture displayed a similar ration of cells following EpCAM-AsiCtargeting PLK1 treatment the ratio of EpCAM+ cells decreased and EpCAM−cells increased. A representative flow cytometry plot (FIG. 2D), thequantification of the experiment analyzed the ratio of GFP+/GFP− cellsin 4 different cell lines (FIG. 2E). (n=4, * p<0.05, p<0.01).

FIGS. 3A-3D demonstrate that human TNBC tissue specifically takes upCy3-EpCAM aptamers. Experimental design; Cy3-EpCAM-AsiC targeting GFP,Alexa647-siRNA-GFP or Alexa647-chol-siRNA-GFP (2 μM of each) were addedto breast cancer and control explants and incubated for 24 h beforetissue was digested with collagenase to a single cell suspension andanalyzed by flow cytometry(FIG. 3A). Tumor biopsies over express EpCAMand cytokeratin, an epithelial cell marker (FIG. 3B) Representativehistograms from one of three independent experiments show that siRNA andchol-siRNA penetrated both tumor and healthy tissue with similarefficacy while EpCAM-AsiC was selectively uptaken by the tumor tissuebiopsy and not by the healthy control tissue sample (FIG. 3C). Theuptake experiment was repeated in tumors from three different patients,each biopsy receive was tested 3 times for each treatment. A summary ofall three patients (FIG. 3D). (n=3, mock, gray EpCAM, red *P<0.05,**P<0.005, t-test CD4-AsiC versus mock treatment).

FIGS. 4A-4C demonstrate that EpCAM AsiC targeting PLK1 specificallyinhibits tumor initiation in Basal A breast cancer cells. Colony assaysof breast cancer cell lines were treated with EpCAM-AsiC targeting PLK1or GFP (4 uM) or paclitaxel (100 nM) for 24 hr and cultured for 8 daysin drug-free medium. Treatment with paclitaxel decreased colonyformation in all cells lines while treatment with EpCAM-AsiC targetingPLK1 only eliminated colony formation in luminal (MCF7) and basal A(HCC1954) cells, treatment with EpCAM-AsiC targeting GFP had no effect(FIG. 4A). The assay was repeated in 3 more cells lines and results werereproducible (FIG. 4B). Sphere formation assay indicated similarresults, EpCAM-AsiC targeting PLK1 decreased the number of spheres onlyin basal A and luminal cells and had no effect on basal B cells (FIG.4C). MB468-luc cells were treated for 24 h with EpCAM-AsiC targetingeither GFP or PLK1 and injected s.c. to the flank of nude mice. Micewere imaged every 5 days for 20 days. Untreated mice and mice treatedwith EpCAM-AsiC targeting GFP, displayed increase in tumor initiationwhile mice injected with cell pretreated with EpCAM-AsiC targeting PLK1has no tumor initiation.

FIGS. 5A-5C demonstrate the selective uptake of Alexa750-EpCAM-AsiCsinto EpCAM+ tumors. FIG. 5A depicts the experimental setup; nude micewere injected with MB468-luc (left flank) and MB231-luc-mCherry (rightflank) cells, 5 days post injection Alexa750 labeled EpCAM-AsiCtargeting GFP (0.5 mg/kg) was injected s.c. in the neck area. The micewere imaged immediately after injection and again after 24, 48 hr and 5days. The Alexa750 labeled EpCAM-AsiC targeting GFP was co-localizedwith the luciferase tumor in MB468-luc tumor (EpCAM+) and not theMB231-luc-mCherry (EpCAM−) tumor. Analysis of 7 mice indicates asignificant increase of Alexa750 in MB468 (EpCAM+) tumors (FIG. 5B).FIG. 5C depicts a graph of Alexa750 uptake rates.

FIGS. 6A-6B demonstrate the EpCAM AsiC targeting PLK1 specificallyinhibits tumor growth in Basal A breast cancer cells. FIG. 6A depictsthe experimental design. Nude mice injected with eitherMB231-luc-mCherry cells (5×10⁵) or MB468-luc cells (5×10⁶) were treatedwith 5 mg/Kg of either EpCAM AsiC targeting PLK1 or GFP every 72 h orleft untreated. FIG. 6B: MB468-luc tumors treated with EpCAM-AsiCtargeting PLK1 shrunk in size as early as 6 days post treatment and inmany mice completely disappeared after 14 days, Untreated tumors bothEpCAM+ and EpCAM-increased in size over the 14 days.

FIG. 7 demonstrates that EpCAM AsiCs are stable in human and mouseserum. eGFP EpCAM-AsiCs synthesized using 2′-fluoro-pyrimidines,chemically-stabilized cholesterol-conjugated eGFP siRNAs (chol-siRNA),or unmodified eGFP siRNAs were incubated with an equal volume of humanor mouse serum. Aliquots were removed at regular intervals andresuspended in gel loading buffer and stored at −80° C. beforeelectrophoresis on denaturing PAGE gels. The average intensity (+S.E.M.)of bands from 2 independent experiments quantified by densitometry afterstaining is shown.

FIGS. 8A-8B demonstrate that injection of EpCAM AsiCs does not stimulateinnate immunity in mice. Mice were injected sc with eGFP EpCAM-AsiCs (5mg/kg, n=3) or ip with Poly(I:C) (5 or 50 mg/kg (n=2/dose). FIG. 8A:Serum samples, collected at baseline and 6 and 16 hr after treatmentwere assessed for IFNβ, IL-6 and IP-10 by multiplex immunoassay. *p<0.05. ** p<0.01, *** p<0.001, compared to baseline. FIG. 8B: mRNAexpression of cytokine and IFN-induced genes, relative to gapdh wasassayed by qRT-PCR in total splenocytes harvested 16 hr post treatment.** p<0.01, compared to untreated (NT, n=3).

FIG. 9 depicts a table of sequences. (SEQ ID NOS 1-2 and 23-32,respectively, in order of appearance).

FIGS. 10A-10B depict aptamers-siRNA chimera (AsiC). FIG. 10A depicts adiagram of the AsiC (aptamer covalently linked to one strand of ansiRNA) specifically recognizing a cancer cell surface receptor, beingendocytosed and then released to the cytosol, where it is processed likeendogenous pre-miRNAs to knockdown a target gene. Bars indicate the 2delivery hurdles—cell uptake and release from endosomes to the cytosolwhere Dicer and the RNA induced silencing complex (RISC) are located.FIG. 10B depicts the design of the EpCAM AsiC targeting PLK1. (sensestrand disclosed as SEQ ID NO: 1 and antisense strand disclosed as SEQID NO: 2).

FIGS. 11A-11D demonstrate that EpCAM-AsiC knockdown and antitumor effectcorrelates with EpCAM levels and inhibits epithelial breast tumor T-ICs.FIGS. 11A-11B: Representative experiment (FIG. 11A) and AKT1 knockdowncomparing EpCAM-AsiC with lipid siRNA transfection (FIG. 11B). FIG. 11C:Anti-proliferative effect of EpCAM-AsiCs knocking down PLK1 only inEpCAM+ cell lines. D PLK1 EpCAM-AsiCs inhibit colony formation inluminal MCF and basal-A TNBC HCC1143, but not in mesenchymal basal-BMB231 cells.

FIGS. 12A-12B demonstrate the identification of a functional EphA2aptamer FIG. 12A:Incubation of EphA2+ basal-B MB231 cells with EphA2aptamer (EphA2apt) leads to EphA2 degradation and a transient decreasein active Akt (pAkt). FIG. 12B: EphA2+ breast cancer cells incubated for2 h with EphA2apt (0 to 100 nM), but not control nonbinding aptamer(ctl), show reduced EphA2. Addition of Ephrin A was used as a positivecontrol for EphA2 degradation.

FIGS. 13A-13C EpCAM-AsiCs knockdown GFP protein (FIG. 13A) and AKT1 mRNA(FIGS. 13B-13C) only in EpCAM+ cell lines, but not in immortalizedbreast epithelial cell line (BPE) or mesenchymal basal B TNBC or humanfibroblasts. A transfected siRNA is nonspecific in its knockdown. *,P<0.05

FIG. 14. Normal breast tissue and basal-A TNBC tumor biopsies from thesame subject were incubated with Cy3-labeled EpCAM-AsiC and single cellsuspensions were analyzed 3 d later for uptake by flow cytometry. NakedsiRNAs were not taken up by either, cholesterol-conjugated siRNAs wereequally taken up, but EpCAM-AsiCs were specifically taken up by thetumor. Representative tissues are shown at left.

FIGS. 15A-15C. Treatment of EpCAM+, but with not EpCAM−, breast cancerlines with PLK1 EpCAM-AsiCs inhibits colony (FIGS. 15A, 15B) andmammosphere (FIG. 15C) function, in vitro assays of T-IC function.

FIG. 16 demonstrates that ex vivo treatment of MB468 cells with PLK1EpCAM-AsiCs eliminated their ability to form tumors in nude mice. Anequal number of viable cells were implanted the day after treatment.

FIGS. 17A-17B demonstrate that EpCAM-AsiCs are selectively taken up intoEpCAM+, but not EpCAM−, TNBC tumors. FIG. 17A depicts the experimentalscheme. FIG. 17B depicts the concentration of EpCAM-AsiCs in excisedtumors at sacrifice.

FIG. 18A-18B demonstrate that PLK1 EpCAM-AsiCs caused complete tumorregression of EpCAM+ TNBC xenografts, but had no effect on EpCAM−basal-B xenografts. FIG. 18A depicts the experimental design. Imaging ofluciferase activity of left and right flank tumors was performedsequentially over 2 wks. FIG. 18B depicts a graph of tumor size byluciferase activity. All the EpCAM+ tumors in mice treated with PLK1AsiCs rapidly regressed, while the other tumors continued to grow.

FIGS. 19A-19C demonstrate that basal dependency genes include 4tri-snRNP spliceosome complex genes (PFPF8, PRPF38A, RBM22, USP39), 2nuclear export genes (NUP205, RAN), and a kinetochore gene (NDC80). FIG.19A depicts cell viability, 3 d after knockdown, normalized to controlsiRNA. FIG. 19B depicts colony formation assessed by plating viablecells 2 d after knockdown. FIG. 19C depicts caspase activation 2 d afterknockdown is specific for MB468 and does not occur in BPE cells.

FIG. 20 depicts some possible designs for multimerized EpCAM-AsiCs toimprove endocytosis. In these designs the sense and antisense strandscould be exchanged and the linkers could be varied.

FIGS. 21A-21D demonstrate that EpCAM aptamer specifically targets BasalA breast cancer cells. FIG. 21A depicts the design of EpCAM-AsiC,containing an EpCAM aptamer and a PLK1 siRNA (sense strand disclosed asSEQ ID NO: 1 and antisense strand disclosed as SEQ ID NO: 2). FIG. 21Bdepicts graphs demonstrateing that epithelial breast cancer cell line(BPLER) over express EpCAM protein compared to normal breast epithelialcell line (BPE). EpCAM-AsiC targeting GFP was Alexa647 or Cy3 labeled atthe 3′ end of the antisense siRNA strand and incubated with BPLER andBPE cells. Uptake was assessed 24 hours later by flow cytometry (FIG.21C). Data are representative of 3 independent experiments. Cy3 andAlexa647-labeled EpCAM-AsiC was taken up by MB468 and BPLER (EpCAM+cells) respectively and not by BPE (EpCAM−). MFI of each peak is shown(mock, gray). FIG. 21D depicts graphs of experiments in which, to testfor gene silencing, BPLER and BPE were treated with EpCAM-AsiC targetingGFP (4 μM) and compared to Transfection controls using Dharmafect andGFP-siRNA (100 nM). Knockdown was assessed by flow cytometry 72 hoursafter incubation. Controls were mock and Dhrmafect only treatment(lipid). (n=4).

FIG. 22 depicts graphs demonstrating that EpCAM aptamers do not bindmouse EpCAM. Mouse ESA (EpCAM) levels were determined using flowcytometry with a mCD326 antibody. 4T1 cell an epithelial mouse breastcancer cell line displayed high expression levels of EpCAM. Both RAW(mouse monocyte cell line) and MB468 (human basal A cell line) displayedan increase in EpCAM expression but much smaller than 4T1 cells. A mousemesanchymal cancer cell line (67NR) displayed a minimal increase inEpCAM expression. Uptake experiments demonstrated that EpCAM-Aptamer wasnot taken up by neither 4T1 nor 67NR cells.

FIG. 23 depicts graphs demonstrating that EpCAM is over expressed inbasal A and luminal but not basal B breast cancer cell lines.Representative FACS plots of 8 different breast cancer cell lines,testing EpCAM expression levels by flow cytometery using a hEpCAMAntibody. EpCAM is over expressed in all basal A and luminal cells linesand not in basal B. (mock, shaded gray EpCAM, black)

FIGS. 24A-24F demonstrate that EpCAM AsiC specifically silences geneexpression in Basal A breast cancer cells. EpCAM-AsiC targeting AKT1selectively knocks-down AKT1 mRNA (FIG. 24A) and protein (FIGS. 24B,24C) expression in basal A and luminal breast cancer cell lines and notin basal B or human fibroblasts (hFb). Transfection with siRNA targetingAKT1 induces gene knockdown in all cell lines, while treatment withEpCAM-AsiC targeting GFP doesn't effect AKT1 mRNA and protein levels (*p<0.05, p<0.01). Plots of AKT1 Protein and gene Knockdown comparing theeffect of EpCAM-AsiC to siRNA transfection. EpCAM-AsiC induced knockdowncorrelates with EpCAM expression (FIG. 24D, 24E). (n=3; mean±SEMnormalized to mock; *P<0.05, **P<0.01, 2-tailed t test). FIG. 24Fdepicts the results of flow cytometry analysis.

FIGS. 25A-25E demonstrate that human TNBC tissue specifically takes upCy3-EpCAM aptamers. FIG. 25A depicts the experimental design;Cy3-EpCAM-AsiC targeting GFP, Alexa647-siRNA-GFP orAlexa647-chol-siRNA-GFP (2 μM of each) were added to breast cancer andcontrol explants and incubated for 24 h before tissue was digested withcollagenase to a single cell suspension and analyzed by flow cytometry.FIG. 25B depicts graphs demonstrating that tumor biopsies over expressEpCAM and cytokeratin, an epithelial cell marker. FIG. 25C depictsrepresentative histograms from one of three independent experiments showthat siRNA and chol-siRNA penetrated both tumor and healthy tissue withsimilar efficacy while EpCAM-AsiC was selectively uptaken by the tumortissue biopsy and not by the healthy control tissue sample. The uptakeexperiment was repeated in tumors from three different patients, eachbiopsy received was tested 3 times for each treatment. FIG. 25D depictsrepresentative tumors. A summary of all three patients is depicted inFIG. 25E. (n=3, *P<0.05, **P<0.005, t-test CD4-AsiC versus mocktreatment).

FIG. 26 depicts graphs demonstrating that EpCAM-AsiC is taken up by bothhealthy and colon cancer biopsies. Cy3-EpCAM-AsiC targeting GFP,Alexa647-siRNA-GFP or Alexa647-chol-siRNA-GFP (2 μM of each) were addedto colon cancer and control explants and incubated for 24 h beforetissues were digested with collagenase to a single cell suspension andanalyzed by flow cytometry. Representative histograms show thatEpCAM-AsiC, siRNA and chol-siRNA penetrated both tumor and healthytissue with similar efficacy.

FIGS. 27A-27D demonstrate that EpCAM AsiC targeting PLK1 specificallyinhibits cell proliferation in Basal A breast cancer cells. The effectof EpCAM-AsiC targeting PLK1 on cell proliferation was tested on 10breast cancer cell lines representative of basal A, B and luminal celllines using cell-titer-glo assay (CTG). EpCAM-AsiC targeting PLK1decreased cell proliferation in both basal A and luminal cell lineswhile having no effect on basal B cells (FIG. 27A). A correlation wasseen between EpCAM expression levels and cell viability (FIG. 27B).Basal A (EpCAM+GFP−) cell were co-cultured with BPE (EpCAM-GFP+) cellsand treated with EpCAM-AsiC targeting PLK1 or untreated. Untreatedco-culture displayed a similar ration of cells following EpCAM-AsiCtargeting PLK1 treatment the ratio of EpCAM+ cells decreased and EpCAM−cells increased. FIG. 27C depicts representative flow cytometry plots,and FIG. 27D depicts a graph of the quantification of the experimentanalyzed the ratio of GFP+/GFP− cells in 4 different cell lines. (n=4, *p<0.05, p<0.01).

FIG. 28 depicts a graph demonstrating specific decrease in cellviability in Basal A breast cancer cell lines is PLK1 dependent. Tendifferent breast cancer cell lines representing basal A, B and luminalcells were treated with either EpCAM-AsiC targeting PLK1 or just theEpCAM-aptamer and compared to untreated controls. None of the cell linestreated with EpCAM-aptamer displayed decrease in cell viability, whilebasal A and luminal cell lines displayed a decrease in cell viabilityfollowing treatment with EpCAM-AsiC targeting PLK1.

FIGS. 29A-29C demonstrate that EpCAM AsiC targeting PLK1 specificallyinhibits tumor initiation in Basal A breast cancer cells. Colony assaysof breast cancer cell lines were treated with EpCAM-AsiC targeting PLK1or GFP (4 uM) or paclitaxel (100 nM) for 24 hr and cultured for 8 daysin drug-free medium. Treatment with paclitaxel decreased colonyformation in all cells lines while treatment with EpCAM-AsiC targetingPLK1 only eliminated colony formation in luminal (MCF7) and basal A(HCC1954) cells, treatment with EpCAM-AsiC targeting GFP had no effect.FIG. 29A depicts images of the assay results. The assay was repeated in3 more cells lines and results were reproducible, as demonstrated in thegraph depicted in FIG. 29B. FIG. 29C depicts a graph demonstrating thatsphere formation assay indicated similar results, EpCAM-AsiC targetingPLK1 decreased the number of spheres only in basal A and luminal cellsand had no effect on basal B cells. MB468-luc cells were treated for 24h with EpCAM-AsiC targeting either GFP or PLK1 and injected s.c. to theflank of nude mice. Mice were imaged every 5 days for 20 days. Untreatedmice and mice treated with EpCAM-AsiC targeting GFP, displayed increasein tumor initiation while mice injected with cell pretreated withEpCAM-AsiC targeting PLK1 has no tumor initiation.

FIGS. 30A-30B demonstrate that EpCAM AsiC is stable in human and mouseserum for 36 hours. EpCAM-AsiC targeting GFP synthesized using2′-fluoro-pyrimidines, chemically-stabilized 21-mercholesterol-conjugated GFP-siRNAs (chol-siRNA), and unmodified 21-merGFP-siRNA, each in 100 ul PBS, which were added to 100 μl of of human ormouse serum. At regular intervals, 20 μL was removed, and resuspended ingel loading buffer and frozen at −80° C. before being electrophoresed ona denaturing PAGE gel. FIG. 30A depicts representative PAGE gels andFIG. 30B depicts graphs of the average intensity (+S.E.M.) of bands fromtwo independent experiments analyzed by densitometry. Both thestabilized cholesterol-conjugated siRNA and the EpCAM-AsiC are stableover the 36 h of the experiment.

FIGS. 31A-31B demonstrate selective uptake of Alexa750-EpCAM-AsiCs intoEpCAM+ tumors. FIG. 31A depicts the experimental setup; nude mice wereinjected with MB468-luc (left flank) and MB231-luc-mCherry (right flank)cells, 5 days post injection Alexa750 labeled EpCAM-AsiC targeting GFP(0.5 mg/kg) was injected s.c. in the neck area. The mice were imagedimmediately after injection and again after 24, 48 hr and 5 days. TheAlexa750 labeled EpCAM-AsiC targeting GFP was co-localized with theluciferase tumor in MB468-luc tumor (EpCAM+) and not theMB231-luc-mCherry (EpCAM−) tumor. FIG. 31B depicts a graph of analysisof 7 mice indicating a significant increase of Alexa750 in MB468(EpCAM+) tumors. At day 5 the tumors were removed and visualized tovalidate that the Alexa750 labeled EpCAM-AsiC targeting GFP indeedentered the tumors. Increased level of Alexa750 is negatively correlatedwith mCherry levels. (n=8, *P<0.05, t-test EpCAM+ versus EpCAM− cells).

FIGS. 32A-32B demonstrate that EpCAM AsiC targeting PLK1 specificallyinhibits tumor growth in Basal A breast cancer cells. FIG. 32A depictsthe experimental setup; nude mice injected with either MB231-luc-mCherrycells (5×10⁵) or MB468-luc cells (5×10⁶) were treated with 5 mg/Kg ofeither EpCAM AsiC targeting PLK1 or GFP every 72 h or left untreated.Mice were imaged using the IVIS Spectra imaging system every 72 h for 14days. FIG. 32B depicts a graph demonstrating that MB468-luc tumorstreated with EpCAM-AsiC targeting PLK1 shrunk in size as early as 6 dayspost treatment and in many mice completely disappeared after 14 days,Untreated tumors both EpCAM+ and EpCAM− increased in size over the 14days.

FIG. 33 depicts graphs of tumor growth demonstrating that MB468 tumorsregress only after treatment with PLK1 EpCAM-AsiC. Mice with sc MB468tumors were treated with 5 mg/kg RNA 2×/wk beginning when tumors becamepalpable. PLK1 EpCAM-AsiC, GFP SpCAM-AsiC, EpCAM aptamer, PLK1 siRNA,and mock treated samples were analyzed as indicated.

FIG. 34 demonstrates that PLK1 siRNA associates with Argonaute (AGO) incells treated with PLK1 EpCAM-AsiCs. MB-468 cells, treated with PLK1EPCAM-AsiC or siRNA for 2 days, were lysed, and cell lysates wereimmunoprecipitated with pan-AGO antibody or IgG isotype control. Theamount of PLK1 siRNA in the immunoprecipitates was quantified by TaqmanqRT-PCR, presented as log₂ mean with SEM, relative to miR-16. **, P<0.01by Student's t-test relative to siRNA-treated cells. ND, not detectable.PLK1 siRNA was found in the RISC after treatment with PLK1 EpCAM-AsiCs.However, the Ago immunoprecipitation did not significantly deplete PLK1siRNAs from the supernatant. This is likely because most RNAs that aretaken up by cells are not released from endosomes to the cytosol (A.Wittrup et al., Visualizing lipid-formulated siRNA release fromendosomes and target gene knockdown. Nature Biotechnology 2015, inpress).

FIG. 35 demonstrates that PLK EpCAM AsiC suppresses MCF10CA1a (CA1a)tumor growth. The top panel depicts the experimental scheme. In thisexperiment the AsiCs were injected sc in the flank near the tumor, butnot into the tumor. The bottom panel depicts a graph of Log₂ totalluminescent photon flux of the tumors (N=4); *, P<0.05 by Student'st-test.

DETAILED DESCRIPTION

The inventors have demonstrated the suprising efficacy of AsiCs(aptamer-siRNA chimeric molecules) in treating cancer. The AsiC'sdescribed herein utilize an aptamer that targets the chimeric moleculespecifically to cancer cells, providing effective and on-targetsuppression of the gene targeted by the siRNA.

In particular, the aptamers described herein, e.g. those targeting EpCAMand EphA2, permit the therapy to target tumor-initiating cells (alsoreferred to as cancer stem cells). These cells are responsible not onlyfor tumor initiation, replapse, and metastasis, but are also relativelyresistant to conventional cytotoxic therapy. Thus, the compositions andmethods described herein permit effective treatment of the underlyingpathology in a way that existing therapies fail to do. The success ofthe AsiC's described herein is particularly suprising in that directtargeting of EpCAM with antibodies has been previously investigated andfound to lack effectiveness.

Moreover, the AsiC's described herein are demonstrated to besurprisingly efficacious in the treatment of epithelial cancers, e.g.breast cancer (e.g. triple negative breast cancer (TNBC)). There are nocurrent targeted therapies for TNBC and what treatments are availabletypically result in metastasis within 3 years, leading to death. TheAsiC's described herein demonstrated effective gene knockdownspecifically in luminal and basal-A TNBC cells as compared to healthycells, suppressed colony and mammosphere formation in vitro andabrogated tumor initiation ex vivo. In vitro treatment with the AsiC'sresulted in targeted delivery of the therapeutic and rapid tumorregression.

In one aspect, described herein is a chimeric molecule comprising acancer marker-binding domain and an inhibitory nucleic acid domain. Asused herein, “cancer marker-binding domain” refers to a domain and/ormolecule that can bind specifically to a molecule more highly expressedon the surface of a cancer cell as compared to a healthy cell of thesame type (a cancer marker). In some embodiments, the cancer marker canbe a protein and/or polypeptide. In some embodiments, the cancer markercan be selected from EpCAM or EphA2. In some embodiments, the cancermarker-binding domain can be an aptamer.

As used herein, “EpCAM” or “epithelial cell adhesion molecule” refers toa transmembrane glycoprotein mediating Ca2+-independent homotypiccell-cell adhesion in epithelial cells. Sequences for EpCAM are knownfor a variety of species, e.g., human EpCAM (see, e.g., NCBI GeneID:4072; protein sequence: NCBI Ref Seq: NP_002345.2).

As used herein, “EphA2” or “EPH receptor A2” refers to a ephirin typeprotein-tyrosine kinase receptor. EphA2 binding ephrin-A ligands andpermits entry of Kaposi sarcoma-associated herpesvirus into host cells.Sequences for EphA2 are known for a variety of species, e.g., humanEphA2 (see, e.g., NCBI Gene ID: 1969; protein sequence: NCBI Ref Seq:NP_004422.2).

As used herein, “inhibitory nucleic acid domain” refers to a domaincomprising an inhibitory nucleic acid. In some embodiments, theinhibitory nucleic acid can be a siRNA.

The inhibitory nucleic acid domain can inhibit, e.g., can target, theexpression of a gene product that is upregulated in a cancer cell and/orthe expression of a gene that is required for cell growth and/orsurvival. In some embodiments, the inhibitory nucleic acid domain caninhibit the expression of a gene selected from Plk1 (e.g. “polo-likekinase 1”; NCBI Gene ID: 5347); MCL1 (e.g. myeloid cell leukemia 1; NCBIGene ID: 4170); EphA2 (NCBI Gene ID: 1969); PsmA2 (e.g. proteasomesubunit alpha 2; NCBI Gene ID: 5683); MSI1 (e.g., musashi RNA-bindingprotein 1; NCBI Gene ID: 4440); BMI1 (e.g., B lymphoma Mo-MLV insertion1, NCBI Gene ID: 648); XBP1 (X-boxn binding protein 1; NCBI Gene ID:7494); PRPF8 (e.g., pre-mRNA processing factor 8; NCBI Gene ID:10594),PFPF38A (e.g., pre-mRNA processing factor 38A; NCBI Gene ID: 84950),RBM22 (e.g., RNA binding motif protein 22; NCBI Gene ID: 55696), USP39(e.g., ubiquitin specific peptidase 39; NCBI Gene ID: 10713); RAN (e.g.,ras-related nuclear protein; NCBI Gene ID: 5901); NUP205 (e.g.,nucleoporin 205 kDa; NCBI Gene ID: 23165), and NDC80 (e.g., NDC80kinetochore complex component; NCBI Gene ID: 10403). Sequences of thesegenes, e.g., the human mRNAs, are readily obtained from the NCBIdatabase and can be used by one of skill in the art to design inhibitorynucleic acids. Furthermore, provided herein are exemplary inhibitorynucleic acid domains, e.g. a nucleic acid having the sequence of SEQ IDNO: 2.

In some embodiments, a composition as described herein can comprise acancer marker-binding domain comprising an aptamer and an inhibitorynucleic acid domain comprising an siRNA, e.g. the composition cancomprise an aptamer-siRNA chimera (AsiC).

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having cancer with a composition asdescribed herein. Subjects having cancer can be identified by aphysician using current methods of diagnosing cancer. Symptoms and/orcomplications of cancer which characterize these conditions and aid indiagnosis are well known in the art and include but are not limited to,for example, in the case of breast cancer a lump or mass in the breasttissue, swelling of all or part of a breast, skin irritation, dimplingof the breast, pain in the breast or nipple, nipple retraction, redness,scaliness, or irritation of the breast or nipple, and nipple discharge.Tests that may aid in a diagnosis of, e.g. breast cancer include, butare not limited to, mammograms, x-rays, MRI, ultrasound, ductogram, abiopsy, and ductal lavage. A family history of cancer or exposure torisk factors for cancer (e.g. smoke, radiation, pollutants, BRCA1mutation, etc.) can also aid in determining if a subject is likely tohave cancer or in making a diagnosis of cancer.

The terms “malignancy,” “malignant condition,” “cancer,” or “tumor,” asused herein, refer to an uncontrolled growth of cells which interfereswith the normal functioning of the bodily organs and systems.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues. Cancer cells can also spread to otherparts of the body through the blood and lymph systems.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancer cells form tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancer cellsthat form tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably.

A subject that has a cancer or a tumor is a subject having objectivelymeasurable cancer cells present in the subject's body. Included in thisdefinition are malignant, actively proliferative cancers, as well aspotentially dormant tumors or micrometastatses. Cancers which migratefrom their original location and seed other vital organs can eventuallylead to the death of the subject through the functional deterioration ofthe affected organs. Hemopoietic cancers, such as leukemia, are able toout-compete the normal hemopoietic compartments in a subject, therebyleading to hemopoietic failure (in the form of anemia, thrombocytopeniaand neutropenia) ultimately causing death.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.In some embodiments, the cancer can be epithelial cancer. In someembodiments, the cancer can be breast cancer. In some embodiments, thecancer can be triple negative breast cancer.

A “cancer cell” is a cancerous, pre-cancerous, or transformed cell,either in vivo, ex vivo, or in tissue culture, that has spontaneous orinduced phenotypic changes that do not necessarily involve the uptake ofnew genetic material. Although transformation can arise from infectionwith a transforming virus and incorporation of new genomic nucleic acid,or uptake of exogenous nucleic acid, it can also arise spontaneously orfollowing exposure to a carcinogen, thereby mutating an endogenous gene.Transformation/cancer is associated with, e.g., morphological changes,immortalization of cells, aberrant growth control, foci formation,anchorage independence, malignancy, loss of contact inhibition anddensity limitation of growth, growth factor or serum independence, tumorspecific markers, invasiveness or metastasis, and tumor growth insuitable animal hosts such as nude mice. See, e.g., Freshney, CULTUREANIMAL CELLS: MANUAL BASIC TECH. (3rd ed., 1994).

The compositions and methods described herein can be administered to asubject having or diagnosed as having cancer. In some embodiments, themethods described herein comprise administering an effective amount ofcompositions described herein, to a subject in order to alleviate asymptom of a cancer. As used herein, “alleviating a symptom of a cancer”is ameliorating any condition or symptom associated with the cancer. Ascompared with an equivalent untreated control, such reduction is by atleast 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more asmeasured by any standard technique. A variety of means for administeringthe compositions described herein to subjects are known to those ofskill in the art. Such methods can include, but are not limited to oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, topical, injection, orintratumoral administration. Administration can be local or systemic. Insome embodiments, the administration is subcutaneous. In someembodiments, the administration of an AsiC as described herein issubcutaneous.

The term “effective amount” as used herein refers to the amount of of acomposition needed to alleviate at least one or more symptom of thedisease or disorder, and relates to a sufficient amount ofpharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount that issufficient to provide a particular anti-cancer effect when administeredto a typical subject. An effective amount as used herein, in variouscontexts, would also include an amount sufficient to delay thedevelopment of a symptom of the disease, alter the course of a symptomdisease (for example but not limited to, slowing the progression of asymptom of the disease), or reverse a symptom of the disease. Thus, itis not generally practicable to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” can bedetermined by one of ordinary skill in the art using only routineexperimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of a composition) which achieves a half-maximal inhibitionof symptoms) as determined in cell culture, or in an appropriate animalmodel. Levels in plasma can be measured, for example, by highperformance liquid chromatography. The effects of any particular dosagecan be monitored by a suitable bioassay, e.g., assay for tumor size,among others. The dosage can be determined by a physician and adjusted,as necessary, to suit observed effects of the treatment.

In some embodiments, the technology described herein relates to apharmaceutical composition as described herein, and optionally apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers and diluents include saline, aqueous buffer solutions, solventsand/or dispersion media. The use of such carriers and diluents is wellknown in the art. Some non-limiting examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent,e.g. as described herein.

In some embodiments, the pharmaceutical composition as described hereincan be a parenteral dose form. Since administration of parenteral dosageforms typically bypasses the patient's natural defenses againstcontaminants, parenteral dosage forms are preferably sterile or capableof being sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. In addition, controlled-release parenteraldosage forms can be prepared for administration of a patient, including,but not limited to, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms asdisclosed within are well known to those skilled in the art. Examplesinclude, without limitation: sterile water; water for injection USP;saline solution; glucose solution; aqueous vehicles such as but notlimited to, sodium chloride injection, Ringer's injection, dextroseInjection, dextrose and sodium chloride injection, and lactated Ringer'sinjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and propylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.Compounds that alter or modify the solubility of a pharmaceuticallyacceptable salt can also be incorporated into the parenteral dosageforms of the disclosure, including conventional and controlled-releaseparenteral dosage forms.

Pharmaceutical compositions can also be formulated to be suitable fororal administration, for example as discrete dosage forms, such as, butnot limited to, tablets (including without limitation scored or coatedtablets), pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion. Such compositions contain a predetermined amountof the pharmaceutically acceptable salt of the disclosed compounds, andmay be prepared by methods of pharmacy well known to those skilled inthe art. See generally, Remington: The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the composition can be administered in asustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

The methods described herein can further comprise administering a secondagent and/or treatment to the subject, e.g. as part of a combinatorialtherapy. Non-limiting examples of a second agent and/or treatment caninclude radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel,carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide,rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaI1l (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE™. vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(Camptosar, CPT-11) (including the treatment regimen of irinotecan with5-FU and leucovorin); topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; combretastatin; leucovorin (LV); oxaliplatin, includingthe oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb™);inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®))and VEGF-A that reduce cell proliferation and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

In addition, the methods of treatment can further include the use ofradiation or radiation therapy. Further, the methods of treatment canfurther include the use of surgical treatments.

In some embodiments of any of the aspects described herein, a chimericmolecule as described herein can be administered in combination with ataxane (e.g. docetaxel or paclitaxel). In some embodiments of any of theaspects described herein, a chimeric molecule as described herein can beadministered in combination with paclitaxel. In some embodiments of anyof the aspects described herein, an AsiC as described herein can beadministered in combination with a taxane. In some embodiments of any ofthe aspects described herein, an AsiC as described herein can beadministered in combination with paclitaxel.

In certain embodiments, an effective dose of a composition as describedherein can be administered to a patient once. In certain embodiments, aneffective dose of a composition can be administered to a patientrepeatedly. For systemic administration, subjects can be administered atherapeutic amount of a composition comprising such as, e.g. 0.1 mg/kg,0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg,20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatmentscan be administered on a less frequent basis. For example, aftertreatment biweekly for three months, treatment can be repeated once permonth, for six months or a year or longer. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80% or at least 90% or more.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to the composition. Thedesired dose or amount of activation can be administered at one time ordivided into subdoses, e.g., 2-4 subdoses and administered over a periodof time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more. Acomposition can be administered over a period of time, such as over a 5minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of cancer.A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. cancer) or one or more complications related to such a condition,and optionally, have already undergone treatment for cancer or the oneor more complications related to cancer. Alternatively, a subject canalso be one who has not been previously diagnosed as having cancer orone or more complications related to cancer. For example, a subject canbe one who exhibits one or more risk factors for cancer or one or morecomplications related to cancer or a subject who does not exhibit riskfactors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

Inhibitors of the expression of a given gene can be an inhibitorynucleic acid or inhibitory oligonucleotide. In some embodiments, theinhibitory nucleic acid is an inhibitory RNA (iRNA). In someembodiments, the inhibitory nucleic acid is an inhibitory DNA (iDNA).Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). The inhibitory nucleic acids described herein caninclude an RNA or DNA strand (the antisense strand) having a regionwhich is 30 nucleotides or less in length, i.e., 8-30 nucleotides inlength, generally 19-24 nucleotides in length, which region issubstantially complementary to at least part of a precursor or matureform of a target gene's transcript. The use of these inhibitoryoligonucleotides enables the targeted degradation of the target gene,resulting in decreased expression and/or activity of the target gene.

As used herein, the term “inhibitory oligonucleotide,” “inhibitorynucleic acid,” or “antisense oligonucleotide” (ASO) refers to an agentthat contains an oligonucleotide, e.g. a DNA or RNA molecule whichmediates the targeted cleavage of an RNA transcript. In one embodiment,an inhibitory oligonucleotide as described herein effects inhibition ofthe expression and/or activity of a target gene. Inhibitory nucleicacids useful in the present methods and compositions include antisenseoligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, modifiedbases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids(PNAs), and other oligomeric compounds or oligonucleotide mimetics whichhybridize to at least a portion of the target nucleic acid and modulateits function. In some embodiments, the inhibitory nucleic acids includeantisense RNA, antisense DNA, chimeric antisense oligonucleotides,antisense oligonucleotides comprising modified linkages, interferenceRNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA);small RNA-induced gene activation (RNAa); small activating RNAs(saRNAs), or combinations thereof. For further disclosure regardinginhibitory nucleic acids, please see US2010/0317718 (antisense oligos);US2010/0249052 (double-stranded ribonucleic acid (dsRNA));US2009/0181914 and US2010/0234451 (LNAs); US2007/0191294 (siRNAanalogues); US2008/0249039 (modified siRNA); and WO2010/129746 andWO2010/040112 (inhibitory nucleic acids).

In certain embodiments, contacting a cell with the inhibitor (e.g. aninhibitory oligonucleotide) results in a decrease in the target RNAlevel in a cell by at least about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 99%, up to and including 100% of the target mRNA level foundin the cell without the presence of the inhibitory oligonucleotide.

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein effects inhibition of theexpression and/or activity of the target gene. In one aspect, an RNAinterference agent includes a single stranded RNA that interacts with atarget RNA sequence to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, long double stranded RNA introduced intoplants and invertebrate cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleaves the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, anRNA interference agent relates to a double stranded RNA that promotesthe formation of a RISC complex comprising a single strand of RNA thatguides the complex for cleavage at the target region of a targettranscript to effect silencing of the target gene.

In some embodiments, the inhibitory oligonucleotide can be adouble-stranded nucleic acid (e.g. a dsRNA). A double-stranded nucleicacid includes two nucleic acid strands that are sufficientlycomplementary to hybridize to form a duplex structure under conditionsin which the double-stranded nucleic acid will be used. One strand of adouble-stranded nucleic acid (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA and/or the mature miRNA formed during theexpression of the target gene. The other strand (the sense strand)includes a region that is complementary to the antisense strand, suchthat the two strands hybridize and form a duplex structure when combinedunder suitable conditions. Generally, the duplex structure is between 8and 30 inclusive, more generally between 18 and 25 inclusive, yet moregenerally between 19 and 24 inclusive, and most generally between 19 and21 base pairs in length, inclusive. Similarly, the region ofcomplementarity to the target sequence is between 8 and 30 inclusive,more generally between 18 and 25 inclusive, yet more generally between19 and 24 inclusive, and most generally between 19 and 21 nucleotides inlength, inclusive. In some embodiments, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA and/or miRNA targetis a contiguous sequence of an mRNA target of sufficient length to be asubstrate for antisense-directed cleavage (e.g., cleavage through a RISCpathway). Double-stranded nucleic acids having duplexes as short as 8base pairs can, under some circumstances, mediate antisense-directed RNAcleavage. Most often a target will be at least 15 nucleotides in length,preferably 15-30 nucleotides in length.

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a double-stranded inhibitory nucleic acid,e.g., a duplex region of 8 to 36, e.g., 15-30 base pairs. Thus, in oneembodiment, to the extent that it becomes processed to a functionalduplex of e.g., 15-30 base pairs that targets a desired RNA forcleavage, an inhibitory nucleic acid molecule or complex of inhibitorynucleic acid molecules having a duplex region greater than 30 base pairsis a double-stranded nucleic acid. Thus, an ordinarily skilled artisanwill recognize that in one embodiment, then, a miRNA is a dsRNA. Inanother embodiment, a dsRNA is not a naturally occurring miRNA. Inanother embodiment, an inhibitory nucleic acid agent useful to targetthe target gene expression is not generated in the target cell bycleavage of a larger double-stranded nucleic acid molecule.

While a target sequence is generally 15-30 nucleotides in length, thereis wide variation in the suitability of particular sequences in thisrange for directing cleavage of any given target RNA. When miRNAs aretargeted, the target sequence can be as short as 8 nucleotides,including the “seed” region (e.g. nucleotides 2-8)). Various softwarepackages and the guidelines set out herein provide guidance for theidentification of optimal target sequences for any given gene target,but an empirical approach can also be taken in which a “window” or“mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that mayserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aninhibitory nucleic acid agent, mediate the best inhibition of targetgene expression.

A double-stranded inhibitory nucleic acid as described herein canfurther include one or more single-stranded nucleotide overhangs. Thedouble-stranded inhibitory nucleic acid can be synthesized by standardmethods known in the art as further discussed below, e.g., by use of anautomated DNA synthesizer, such as are commercially available from, forexample, Biosearch, Applied Biosystems, Inc. In one embodiment, theantisense strand of a double-stranded inhibitory nucleic acid has a 1-10nucleotide overhang at the 3′ end and/or the 5′ end. In one embodiment,the sense strand of a double-stranded inhibitory nucleic acid has a 1-10nucleotide overhang at the 3′ end and/or the 5′ end. In one embodiment,at least one end of a double-stranded inhibitory nucleic acid has asingle-stranded nucleotide overhang of 1 to 4, generally 1 or 2nucleotides. Double-stranded inhibitory nucleic acids having at leastone nucleotide overhang have unexpectedly superior inhibitory propertiesrelative to their blunt-ended counterparts.

In another embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of aninhibitory nucleic acid, e.g., a dsRNA. For example, when a 3′-end ofone strand of a double-stranded inhibitory nucleic acid extends beyondthe 5′-end of the other strand, or vice versa, there is a nucleotideoverhang. A double-stranded inhibitory nucleic acid can comprise anoverhang of at least one nucleotide; alternatively the overhang cancomprise at least two nucleotides, at least three nucleotides, at leastfour nucleotides, at least five nucleotides or more. A nucleotideoverhang can comprise or consist of a nucleotide/nucleoside analog,including a deoxynucleotide/nucleoside. The overhang(s) may be on thesense strand, the antisense strand or any combination thereof.Furthermore, the nucleotide(s) of an overhang can be present on the 5′end, 3′ end or both ends of either an antisense or sense strand of adouble-stranded inhibitory nucleic acid.

The terms “blunt” or “blunt ended” as used herein in reference to adouble-stranded inhibitory nucleic acid mean that there are no unpairednucleotides or nucleotide analogs at a given terminal end of a dsRNA,i.e., no nucleotide overhang. One or both ends of a double-strandedinhibitory nucleic acid can be blunt. Where both ends of adouble-stranded inhibitory nucleic acid are blunt, the double-strandedinhibitory nucleic acid is said to be blunt ended. To be clear, a “bluntended” double-stranded inhibitory nucleic acid is a double-strandedinhibitory nucleic acid that is blunt at both ends, i.e., no nucleotideoverhang at either end of the molecule. Most often such a molecule willbe double-stranded over its entire length.

In this aspect, one of the two strands is complementary to the other ofthe two strands, with one of the strands being substantiallycomplementary to a sequence of a the target gene precursor or maturemiRNA. As such, in this aspect, a double-stranded inhibitory nucleicacid will include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand and the second oligonucleotide isdescribed as the corresponding antisense strand of the sense strand. Asdescribed elsewhere herein and as known in the art, the complementarysequences of a double-stranded inhibitory nucleic acid can also becontained as self-complementary regions of a single nucleic acidmolecule, as opposed to being on separate oligonucleotides.

The skilled person is well aware that inhibitory nucleic acid having aduplex structure of between 20 and 23, but specifically 21, base pairshave been hailed as particularly effective in inducingantisense-mediated inhibition (Elbashir et al., EMBO 2001,20:6877-6888). However, others have found that shorter or longerinhibitory nucleic acids can be effective as well.

Further, it is contemplated that for any sequence identified, furtheroptimization could be achieved by systematically either adding orremoving nucleotides to generate longer or shorter sequences and testingthose and sequences generated by walking a window of the longer orshorter size up or down the target RNA from that point. Again, couplingthis approach to generating new candidate targets with testing foreffectiveness of inhibitory nucleic acids based on those targetsequences in an inhibition assay as known in the art or as describedherein can lead to further improvements in the efficiency of inhibition.Further still, such optimized sequences can be adjusted by, e.g., theintroduction of modified nucleotides as described herein or as known inthe art, addition or changes in overhang, or other modifications asknown in the art and/or discussed herein to further optimize themolecule (e.g., increasing serum stability or circulating half-life,increasing thermal stability, enhancing transmembrane delivery,targeting to a particular location or cell type, increasing interactionwith silencing pathway enzymes, increasing release from endosomes, etc.)as an expression inhibitor.

An inhibitory nucleic acid as described herein can contain one or moremismatches to the target sequence. In one embodiment, an inhibitorynucleic acid as described herein contains no more than 3 mismatches. Ifthe antisense strand of the inhibitory nucleic acid contains mismatchesto a target sequence, it is preferable that the area of mismatch not belocated in the center of the region of complementarity. If the antisensestrand of the inhibitory nucleic acid contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide inhibitory nucleicacid agent strand which is complementary to a region of the target geneor a precursor thereof, the strand generally does not contain anymismatch within the central 13 nucleotides. The methods described hereinor methods known in the art can be used to determine whether aninhibitory nucleic acid containing a mismatch to a target sequence iseffective in inhibiting the expression of the target gene. Considerationof the efficacy of inhibitory nucleic acids with mismatches ininhibiting expression of the target gene is important, especially if theparticular region of complementarity in the target gene is known to havepolymorphic sequence variation within the population.

In yet another embodiment, the nucleic acid of an inhibitory nucleicacid, e.g., a dsRNA, is chemically modified to enhance stability orother beneficial characteristics. The nucleic acids featured in theinvention may be synthesized and/or modified by methods well establishedin the art, such as those described in “Current protocols in nucleicacid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons,Inc., New York, N.Y., USA, which is hereby incorporated herein byreference. Modifications include, for example, (a) end modifications,e.g., 5′ end modifications (phosphorylation, conjugation, invertedlinkages, etc.) 3′ end modifications (conjugation, DNA nucleotides,inverted linkages, etc.), (b) base modifications, e.g., replacement withstabilizing bases, destabilizing bases, or bases that base pair with anexpanded repertoire of partners, removal of bases (abasic nucleotides),or conjugated bases, (c) sugar modifications (e.g., at the 2′ positionor 4′ position) or replacement of the sugar, as well as (d) backbonemodifications, including modification or replacement of thephosphodiester linkages. Specific examples of nucleic acid compoundsuseful in the embodiments described herein include, but are not limitedto nucleic acids containing modified backbones or no naturalinternucleoside linkages. Nucleic acids having modified backbonesinclude, among others, those that do not have a phosphorus atom in thebackbone. For the purposes of this specification, and as sometimesreferenced in the art, modified nucleic acids that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In particular embodiments, the modified nucleicacid will have a phosphorus atom in its internucleoside backbone.

Modified backbones can include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, each of which is herein incorporated by reference.

Modified backbones that do not include a phosphorus atom therein havebackbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other nucleic acid mimetics suitable or contemplated for use ininhibitory nucleic acids, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a nucleic acid mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar backbone of a nucleic acid isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found, for example,in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include nucleic acids withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH2-NH-CH2-, —CH2-N(CH3)-O—CH2-[known as amethylene (methylimino) or MMI backbone], —CH2-O—N(CH3)-CH2-,—CH2-N(CH3)-N(CH3)-CH2- and —N(CH3)-CH2-CH2-[wherein the nativephosphodiester backbone is represented as —O—P—O—CH2-] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, theinhibitory nucleic acids featured herein have morpholino backbonestructures of the above-referenced U.S. Pat. No. 5,034,506.

Modified nucleic acids can also contain one or more substituted sugarmoieties. The inhibitory nucleic acids featured herein can include oneof the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH2)nO] mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3,O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 toabout 10. In other embodiments, dsRNAs include one of the following atthe 2′ position: C1 to C10 lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN,CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an inhibitory nucleic acid,or a group for improving the pharmacodynamic properties of an inhibitorynucleic acid, and other substituents having similar properties. In someembodiments, the modification includes a 2′ methoxyethoxy(2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the nucleic acid of an inhibitory nucleicacid, particularly the 3′ position of the sugar on the 3′ terminalnucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminalnucleotide. Inhibitory nucleic acids may also have sugar mimetics suchas cyclobutyl moieties in place of the pentofuranosyl sugar.Representative U.S. patents that teach the preparation of such modifiedsugar structures include, but are not limited to, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference.

An inhibitory nucleic acid can also include nucleobase (often referredto in the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The nucleic acid of an inhibitory nucleic acid can also be modified toinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. Thisstructure effectively “locks” the ribose in the 3′-endo structuralconformation. The addition of locked nucleic acids to siRNAs has beenshown to increase siRNA stability in serum, and to reduce off-targeteffects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447;Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. etal., (2003) Nucleic Acids Research 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, each of which is herein incorporated byreference in its entirety.

Another modification of the nucleic acid of an inhibitory nucleic acidfeatured in the invention involves chemically linking to the nucleicacid one or more ligands, moieties or conjugates that enhance theactivity, cellular distribution, pharmacokinetic properties, or cellularuptake of the inhibitory nucleic acid. Such moieties include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid(Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), athioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an inhibitory nucleic acid agent into which it isincorporated. In preferred embodiments a ligand provides an enhancedaffinity for a selected target, e.g, molecule, cell or cell type,compartment, e.g., a cellular or organ compartment, tissue, organ orregion of the body, as, e.g., compared to a species absent such aligand. Preferred ligands will not take part in duplex pairing in aduplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polylysine (PLL), poly L aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as an hepatopcyteor a macrophage, among others. A targeting group can be a thyrotropin,melanotropin, lectin, glycoprotein, surfactant protein A, Mucincarbohydrate, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, glycosylated polyaminoacids, multivalent galactose,transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid,cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A,biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as ahepatocyte or macrophage. Ligands may also include hormones and hormonereceptors. They can also include non-peptidic species, such as lipids,lectins, carbohydrates, vitamins, cofactors, multivalent lactose,multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosaminemultivalent mannose, or multivalent fucose.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the inhibitory nucleic acid agent into the cell, for example,by disrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

In some embodiments, a ligand attached to an inhibitory nucleic acid asdescribed herein acts as a pharmacokinetic (PK) modulator. As usedherein, a “PK modulator” refers to a pharmacokinetic modulator. PKmodulators include lipophiles, bile acids, steroids, phospholipidanalogues, peptides, protein binding agents, PEG, vitamins etc.Examplary PK modulators include, but are not limited to, cholesterol,fatty acids, cholic acid, lithocholic acid, dialkylglycerides,diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen,vitamin E, biotin etc. Oligonucleotides that comprise a number ofphosphorothioate linkages are also known to bind to serum protein, thusshort oligonucleotides, e.g., oligonucleotides of about 5 bases, 10bases, 15 bases or 20 bases, comprising multiple of phosphorothioatelinkages in the backbaone are also amenable to the present invention asligands (e.g. as PK modulating ligands). In addition, aptamers that bindserum components (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

For macromolecular drugs and hydrophilic drug molecules, which cannoteasily cross bilayer membranes, entrapment in endosomal/lysosomalcompartments of the cell is thought to be the biggest hurdle foreffective delivery to their site of action. A number of approaches andstrategies have been devised to address this problem. For liposomalformulations, the use of fusogenic lipids in the formulation have beenthe most common approach (Singh, R. S., Goncalves, C. et al. (2004). Onthe Gene Delivery Efficacies of pH-Sensitive Cationic Lipids viaEndosomal Protonation. A Chemical Biology Investigation. Chem. Biol. 11,713-723. ). Other components, which exhibit pH-sensitive endosomolyticactivity through protonation and/or pH-induced conformational changes,include charged polymers and peptides. Examples may be found in Hoffman,A. S., Stayton, P. S. et al. (2002). Design of “smart” polymers that candirect intracellular drug delivery. Polymers Adv. Technol. 13, 992-999;Kakudo, Chaki, T., S. et al. (2004). Transferrin-Modified LiposomesEquipped with a pH-Sensitive Fusogenic Peptide: An Artificial Viral-likeDelivery System. Biochemistry 436, 5618-5628; Yessine, M. A. and Leroux,J. C. (2004). Membrane-destabilizing polyanions: interaction with lipidbilayers and endosomal escape of biomacromolecules. Adv. Drug Deliv.Rev. 56, 999-1021; Oliveira, S., van Rooy, I. et al. (2007). Fusogenicpeptides enhance endosomal escape improving inhibitory nucleicacid-induced silencing of oncogenes. Int. J. Pharm. 331, 211-4. Theyhave generally been used in the context of drug delivery systems, suchas liposomes or lipoplexes. For folate receptor-mediated delivery usingliposomal formulations, for instance, a pH-sensitive fusogenic peptidehas been incorporated into the liposomes and shown to enhance theactivity through improving the unloading of drug during the uptakeprocess (Turk, M. J., Reddy, J. A. et al. (2002). Characterization of anovel pH-sensitive peptide that enhances drug release fromfolate-targeted liposomes at endosomal pHs is described in Biochim.Biophys. Acta 1559, 56-68).

In certain embodiments, the endosomolytic components can be polyanionicpeptides or peptidomimetics which show pH-dependent membrane activityand/or fusogenicity. A peptidomimetic can be a small protein-like chaindesigned to mimic a peptide. A peptidomimetic can arise frommodification of an existing peptide in order to alter the molecule'sproperties, or the synthesis of a peptide-like molecule using unnaturalamino acids or their analogs. In certain embodiments, they have improvedstability and/or biological activity when compared to a peptide. Incertain embodiments, the endosomolytic component assumes its activeconformation at endosomal pH (e.g., pH 5-6). The “active” conformationis that conformation in which the endosomolytic component promotes lysisof the endosome and/or transport of the modular composition of theinvention, or its any of its components (e.g., a nucleic acid), from theendosome to the cytoplasm of the cell.

Exemplary endosomolytic components include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component can contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of endosomolytic componentsinclude H2N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO2H (SEQ ID NO: 16);H2N-(AALAEALAEALAEALAEALAEALAAAAGGC)-CO2H (SEQ ID NO: 17); andH2N-(ALEALAEALEALAEA)-CONH2 (SEQ ID NO: 18).

In certain embodiments, more than one endosomolytic component can beincorporated into the inhibitory nucleic acid agent of the invention. Insome embodiments, this will entail incorporating more than one of thesame endosomolytic component into the inhibitory nucleic acid agent. Inother embodiments, this will entail incorporating two or more differentendosomolytic components into inhibitory nucleic acid agent.

These endosomolytic components can mediate endosomal escape by, forexample, changing conformation at endosomal pH. In certain embodiments,the endosomolytic components can exist in a random coil conformation atneutral pH and rearrange to an amphipathic helix at endosomal pH. As aconsequence of this conformational transition, these peptides may insertinto the lipid membrane of the endosome, causing leakage of theendosomal contents into the cytoplasm. Because the conformationaltransition is pH-dependent, the endosomolytic components can displaylittle or no fusogenic activity while circulating in the blood (pH˜7.4). “Fusogenic activity,” as used herein, is defined as that activitywhich results in disruption of a lipid membrane by the endosomolyticcomponent. One example of fusogenic activity is the disruption of theendosomal membrane by the endosomolytic component, leading to endosomallysis or leakage and transport of one or more components of the modularcomposition of the invention (e.g., the nucleic acid) from the endosomeinto the cytoplasm.

Suitable endosomolytic components can be tested and identified by askilled artisan. For example, the ability of a compound to respond to,e.g., change charge depending on, the pH environment can be tested byroutine methods, e.g., in a cellular assay. In certain embodiments, atest compound is combined with or contacted with a cell, and the cell isallowed to internalize the test compound, e.g., by endocytosis. Anendosome preparation can then be made from the contacted cells and theendosome preparation compared to an endosome preparation from controlcells. A change, e.g., a decrease, in the endosome fraction from thecontacted cell vs. the control cell indicates that the test compound canfunction as a fusogenic agent. Alternatively, the contacted cell andcontrol cell can be evaluated, e.g., by microscopy, e.g., by light orelectron microscopy, to determine a difference in the endosomepopulation in the cells. The test compound and/or the endosomes canlabeled, e.g., to quantify endosomal leakage.

In another type of assay, an inhibitory nucleic acid agent describedherein is constructed using one or more test or putative fusogenicagents. The inhibitory nucleic acid agent can be labeled for easyvisulization. The ability of the endosomolytic component to promoteendosomal escape, once the inhibitory nucleic acid agent is taken up bythe cell, can be evaluated, e.g., by preparation of an endosomepreparation, or by microscopy techniques, which enable visualization ofthe labeled inhibitory nucleic acid agent in the cytoplasm of the cell.In certain other embodiments, the inhibition of gene expression, or anyother physiological parameter, may be used as a surrogate marker forendosomal escape.

In other embodiments, circular dichroism spectroscopy can be used toidentify compounds that exhibit a pH-dependent structural transition. Atwo-step assay can also be performed, wherein a first assay evaluatesthe ability of a test compound alone to respond to changes in pH, and asecond assay evaluates the ability of a modular composition thatincludes the test compound to respond to changes in pH.

In one embodiment of the aspects described herein, a ligand or conjugateis a lipid or lipid-based molecule. Such a lipid or lipid-based moleculepreferably binds a serum protein, e.g., human serum albumin (HSA). AnHSA binding ligand allows for distribution of the conjugate to a targettissue, e.g., a non-kidney target tissue of the body. Other moleculesthat can bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, such agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

Peptides suitable for use with the present invention can be a naturalpeptide, e.g., tat or antennopedia peptide, a synthetic peptide, or apeptidomimetic. Furthermore, the peptide can be a modified peptide, forexample peptide can comprise non-peptide or pseudo-peptide linkages, andD-amino acids. A peptidomimetic (also referred to herein as anoligopeptidomimetic) is a molecule capable of folding into a definedthree-dimensional structure similar to a natural peptide. The attachmentof peptide and peptidomimetics to inhibitory nucleic acid agents canaffect pharmacokinetic distribution of the inhibitory nucleic acid, suchas by enhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 19). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 20)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 21)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 22))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit is a cell targeting peptidesuch as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In some embodiments, the inhibitory nucleic acid oligonucleotidesdescribed herein further comprise carbohydrate conjugates. Thecarbohydrate conjugates are advantageous for the in vivo delivery ofnucleic acids, as well as compositions suitable for in vivo therapeuticuse, as described herein. As used herein, “carbohydrate” refers to acompound which is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which may belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which may be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4-9 monosaccharide units),and polysaccharides such as starches, glycogen, cellulose andpolysaccharide gums. Specific monosaccharides include C5 and above(preferably C5-C8) sugars; di- and trisaccharides include sugars havingtwo or three monosaccharide units (preferably C5-C8). In someembodiments, the carbohydrate conjugate further comprises other ligandsuch as, but not limited to, PK modulator, endosomolytic ligand, andcell permeation peptide.

In some embodiments, the conjugates described herein can be attached tothe inhibitory nucleic acid oligonucleotide with various linkers thatcan be cleavable or non cleavable. The term “linker” or “linking group”means an organic moiety that connects two parts of a compound. Linkerstypically comprise a direct bond or an atom such as oxygen or sulfur, aunit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, suchas, but not limited to, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or moremethylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8),C(O), substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where R8 ishydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment,the linker is between 1-24 atoms, preferably 4-24 atoms, preferably 6-18atoms, more preferably 8-18 atoms, and most preferably 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. Further examples ofcleavable linking groups include but are not limited to, redox-cleavablelinking groups (e.g. a disulphide linking group (—S—S—)),phosphate-based cleavable linkage groups, ester-based cleavable linkinggroups, and peptide-based cleavable linking groups. Representative U.S.patents that teach the preparation of RNA conjugates include, but arenot limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297;7,037,646; each of which is herein incorporated by reference.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an inhibitory nucleic acid. The presentinvention also includes inhibitory nucleic acid compounds that arechimeric compounds. “Chimeric” inhibitory nucleic acid compounds or“chimeras,” in the context of this invention, are inhibitory nucleicacid compounds, e.g. dsRNAs, which contain two or more chemicallydistinct regions, each made up of at least one monomer unit, i.e., anucleotide in the case of a dsRNA compound. These inhibitory nucleicacid typically contain at least one region wherein the nucleic acid ismodified so as to confer upon the inhibitory nucleic acid increasedresistance to nuclease degradation, increased cellular uptake, and/orincreased binding affinity for the target nucleic acid. An additionalregion of the inhibitory nucleic acid may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofinhibitory nucleic acid inhibition of gene expression. Consequently,comparable results can often be obtained with shorter inhibitory nucleicacids when chimeric inhibitory nucleic acids are used, compared to,e.g., phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the nucleic acid of an inhibitory nucleic acid canbe modified by a non-ligand group. A number of non-ligand molecules havebeen conjugated to inhibitory nucleic acids in order to enhance theactivity, cellular distribution or cellular uptake of the inhibitorynucleic acid, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such nucleic acidconjugates have been listed above. Typical conjugation protocols involvethe synthesis of an nucleic acid bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with thenucleic acid still bound to the solid support or following cleavage ofthe nucleic acid, in solution phase. Purification of the nucleic acidconjugate by HPLC typically affords the pure conjugate.

The term “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a target molecule, such as a polypeptide. For example, anaptamer of the invention can specifically bind to a target molecule, orto a molecule in a signaling pathway that modulates the expressionand/or activity of a target molecule. The generation and therapeutic useof aptamers are well established in the art. See, e.g., U.S. Pat. No.5,475,096.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments, specific binding can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnontarget entity. A reagent specific for a given target is one thatexhibits specific binding for that target under the conditions of theassay being utilized.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. cancer. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with a cancer. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. These and other changes can be made to the disclosure inlight of the detailed description. All such modifications are intendedto be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A chimeric molecule comprising a cancer marker-binding        aptamer domain and an inhibitory nucleic acid domain.    -   2. The molecule of paragraph 1, wherein the cancer marker is        EpCAM or EphA2.    -   3. The molecule of any of paragraphs 1-2, wherein the molecule        is an aptamer-siRNA chimera (AsiC).    -   4. The molecule of any of paragraphs 1-3, wherein the inhibitory        nucleic acid specifically binds to a gene product upregulated in        a cancer cell.    -   5. The molecule of any of paragraphs 1-4, wherein the inhibitory        nucleic acid inhibits the expression of a gene selected from the        group consisting of: Plk1; MCL1; EphA2; PsmA2; MSI1; BMI1; XBP1;        PRPF8; PFPF38A; RBM22; USP39; RAN; NUP205; and NDC80.    -   6. The molecule of any of paragraphs 1-5, wherein the cancer        marker is EpCAM and the inhibitory nucleic acid domain inhibits        the expression of Plk1.    -   7. The molecule of any of paragraphs 1-6, wherein the cancer        marker-binding aptamer domain comprises the sequence of SEQ ID        NO: 33.    -   8. The molecule of any of paragraphs 1-6, wherein the cancer        marker-binding aptamer domain consists essentially of the        sequence of SEQ ID NO: 33.    -   9. The molecule of any of paragraphs 1-8, wherein the inhibitory        nucleic acid domain comprises the sequence of SEQ ID NO: 2.    -   10. The molecule of any of paragraphs 1-8, wherein the        inhibitory nucleic acid domain consists essentially of the        sequence of SEQ ID NO: 2.    -   11. The molecule of any of paragraphs 1-10, comprising the        sequence of one of SEQ ID NOs: 1-3.    -   12. The molecule of any of paragraphs 1-11, consisting        essentially of the sequence of one of SEQ ID NOs: 1-3.    -   13. The molecule of any of paragraphs 1-12, wherein the 3′ end        of the molecule comprises dTdT.    -   14. The molecule of any of paragraphs 1-13, wherein the molecule        comprises at least one 2′-F pyrimidine.    -   15. A pharmaceutical composition comprising the molecule of any        of paragraphs 1-14 and a pharmaceutically acceptable carrier.    -   16. The composition of paragraph 15, comprising at least two        chimeric molecules of any of paragraphs 1-14, wherein the        chimeric molecules have different aptamer domains or inhibitory        nucleic acid domains.    -   17. The composition of paragraph 16, wherein different apatmer        or inhibitory nucleic acid domains recognize different targets.    -   18. The composition of paragraph 16, wherein different apatmer        or inhibitory nucleic acid domains have sequences and recognize        the same target.    -   19. A method of treating cancer, the method comprising        administering a molecule or composition of any of paragraphs        1-18.    -   20. The method of paragraph 19, wherein the cancer is an        epithelial cancer or breast cancer    -   21. The method of paragraph 20, wherein the breast cancer is        triple-negative breast cancer.    -   22. The method of any of paragraphs 19-21, wherein the        administration is subcutaneous.    -   23. The method of any of paragraphs 19-22, wherein the subject        is further administered an additional cancer treatment.    -   24. The method of paragraph 23, wherein the cancer treatment is        paclitaxel.

EXAMPLES Example 1: Gene Knockdown by EpCAM Aptamer-siRNA ChimerasInhibits Basal-Like Triple Negative Breast Cancers and theirTumor-Initiating Cells

Effective therapeutic strategies for in vivo siRNA delivery to knockdowngenes in cells outside the liver are needed to harness RNA interferencefor treating cancer. EpCAM is a tumor-associated antigen highlyexpressed on common epithelial cancers and their tumor-initiating cells(T-IC, also known as cancer stem cells). It is demonstrated herein thataptamer-siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sensestrand and annealed to the siRNA antisense strand) are selectively takenup and knockdown gene expression in EpCAM+ cancer cells in vitro and inhuman cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony andmammosphere formation (in vitro T-IC assays) and tumor initiation byEpCAM+ luminal and basal-A triple negative breast cancer (TNBC) celllines, but not EpCAM− mesenchymal basal-B TNBCs, in nude mice.Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM+ Her2+ andTNBC tumors and suppress their growth. Thus EpCAM-AsiCs provide anattractive approach for treating epithelial cancer.

Introduction

RNA interference (RNAi) offers the opportunity to treat disease byknocking down disease-causing genes.¹ Recent early phase clinical trialshave shown vigorous (75-95%), sustained (lasting for several weeks or upto several months) and safe knockdown of a handful of gene targets inthe liver using lipid nanoparticle-encapsulated or GalNAc-conjugatedsiRNAs.²⁻⁵ The liver, the body's major filtering organ, traps particlesand, hence, is relatively easy to transfect. The major obstacle toharnessing RNAi for treating most diseases however has yet to besolved—namely efficient delivery of small RNAs and gene knockdown incells beyond the liver. In particular, the delivery roadblock is a majorobstacle to harnessing RNAi to treat cancer.⁶

Triple negative breast cancers (TNBC), a heterogeneous group of poorlydifferentiated cancers defined by the lack of estrogen, progesterone andHer2 receptor expression, has the worst prognosis of any breast cancersubtype.⁷⁻⁹ Most TNBCs have epithelial properties and are classified asbasal-like or belong to the basal-A subtype, although a sizable minorityare mesenchymal (basal-B subtype). TNBC afflicts younger women and isthe subtype associated with BRCA1 genetic mutations. No targeted therapyis available. Although most TNBC patients respond to chemotherapy,within 3 years about a third develop metastases and eventually die. Thusnew approaches are needed.

Described herein is a flexible, targeted platform for gene knockdown andtreatment of basal-like TNBCs that might also be suitable for therapyagainst most of the common (epithelial) cancers. We deliver smallinterfering RNAs (siRNA) into epithelial cancer cells by linking them toan RNA aptamer that binds to EpCAM, the first described tumor antigen, acell surface receptor over-expressed on epithelial cancers, includingbasal-like TNBCs. Aptamer-linked siRNAs, known as aptamer-siRNA chimeras(AsiC), have been used in small animal models to treat prostate cancerand prevent HIV infection.¹⁰⁻¹⁸ We chose EpCAM for targeting basal-likeTNBC because EpCAM is highly expressed on epithelial cancers. A highaffinity EpCAM aptamer was previously identified. {Shigdar, 2011 #17903}EpCAM also marks tumor-initiating cells (T-ICs, also known as cancerstem cells).²⁰⁻²⁷ These cells are thought responsible not only forinitiating tumors, but are also relatively resistant to conventionalcytotoxic therapy and are thought responsible for tumor relapse andmetastasis. Devising therapies to eliminate T-ICs is an important unmetgoal of cancer research.²⁸

In normal epithelia, EpCAM is only weakly expressed on basolateral gapjunctions, where it may not be accessible to drugs.²⁹ In epithelialcancers it is not only more abundant (by orders of magnitude), but isalso distributed along the cell membrane. Ligation of EpCAM promotesadhesion and enhances cell proliferation and invasivity. Proteolyticcleavage of EpCAM releases an intracellular fragment that increases stemcell factor transcription.^(30,31) EpCAM's oncogenic properties may makeit difficult for tumor cells to develop resistance by down-modulatingEpCAM. In one study about ⅔ of TNBCs, presumably the basal-A subtype,stained strongly for EpCAM.²⁵ The number of EpCAM+ circulating cells islinked to poor prognosis in breast cancer.³²⁻³⁶ An EpCAM antibody hasbeen evaluated clinically for epithelial cancers, but had limitedeffectiveness on its own.³⁷⁻³⁹ EpCAM expression identifies circulatingtumor cells in an FDA-approved method for monitoring metastatic breast,colon and prostate cancer treatment³²⁻³⁶. Moreover, about 97% of humanbreast cancers and virtually 100% of other common epithelial cancers,including lung, colon, pancreas and prostate, stain brightly for EpCAM,suggesting that the platform developed here could be adapted forRNAi-based therapy of common cancers.

It is demonstrated herein that all epithelial breast cancer cell linestested stained brightly for EpCAM, while immortalized normal breastepithelial cells, fibroblasts and mesenchymal tumor cell lines did not.EpCAM-AsiCs caused targeted gene knockdown in luminal and basal-A TNBCcancer cells and human breast cancer tissues in vitro, but not in normalepithelial cells, mesenchymal tumor cells or normal human breasttissues. Knockdown was proportional to EpCAM expression. MoreoverEpCAM-AsiC-mediated knockdown of PLK1, a gene required for mitosis,suppressed in vitro T-IC functional assays (colony and mammosphereformation) of epithelial breast cancer lines. Ex vivo treatmentspecifically abrogated tumor initiation. Subcutaneously injected PLK1EpCAM-AsiCs were taken up specifically by EpCAM+ basal-A triple negativebreast cancer (TNBC) orthotopic xenografts of poor prognosis basal-A andHer2 breast cancers and caused rapid tumor regression.

EpCAM is Highly Expressed on Epithelial Breast Cancer Cell Lines

First, EpCAM expression was examined in breast cancer cell lines. Basedon gene expression data in the Cancer Cell Line Encyclopedia40,EpCAMmRNA is highly expressed in basal-A TNBC and luminal breast cancer celllines, but poorly in basal-B (mesenchymal) TNBCs (FIG. 1A). SurfaceEpCAM staining, assessed by flow cytometry, was 2-3 logs brighter in allluminal and basal-like cell lines tested, than in normal epitheliaimmortalized with hTERT (BPE)⁴¹, fibroblasts or mesenchymal TNBCs (FIG.1B). Thus EpCAM is highly expressed in epithelial breast cancer celllines compared to normal cells or mesenchymal tumors.

EpCAM-AsiCs Selectively Knock Down Gene Expression in EpCAM+ BreastCancer Cells

A 19 nucleotide (nt) aptamer that binds to human EpCAM with 12 nMaffinity¹⁹ was identified by SELEX.^(42,43) It does not bind to mouseEpCAM (data not shown). A handful of EpCAM-AsiCs that linked either thesense or antisense strand of the siRNA to the 3′-end of the aptamer byseveral linkers were designed and synthesized with 2′-fluoropyrimidinesubstitutions and 3′-dTdT overhangs to enhance in vivo stability, avoidoff-target knockdown of partially complementary genes bearing similarsequences, and limit innate immune receptor stimulation. To test RNAdelivery, gene knockdown and anti-tumor effects, siRNAs wereincorporated to knockdown eGFP (as a useful marker gene); AKT1, anendogenous gene expressed in all the cell lines studied, whose knockdownis not lethal; and PLK1, a kinase required for mitosis, whose knockdownis lethal (FIG. 9). The AsiC that performed best in dose responsestudies of gene knockdown joined the 19 nt EpCAM aptamer to the sense(inactive) strand of the siRNA via a U-U-U linker (FIG. 1C). TheEpCAM-AsiC was produced by annealing the chemically synthesized ˜42-44nt long strand (19 nt aptamer+linker+20-22 nt siRNA sense strand) to a20-22 nt antisense siRNA strand. Commercially synthesized with2′-fluoropyrimidines {Jackson, 2003 #11353; Scacheri, 2004 #11912;Jackson, 2006 #13758; Wheeler, 2011 #17906}, these are RNase resistantand very stable in human serum (T_(1/2)>>36 hr, FIG. 7) and do nottrigger innate immunity when injected in vivo into tumor-bearing mice(FIG. 7).

To verify selective uptake by EpCAM+ tumor cells, confocal fluorescencemicroscopy was used to compare internalization of the EpCAM aptamer,fluorescently labeled at the 5′-end with Cy3, in EpCAM+ MDA-MB-468 TNBCcells and BPE, EpCAM^(dim) immortalized breast epithelial cells (datanot shown). Without wishing to be bound by theory, because AsiCs containonly one aptamer, they do not crosslink the receptor they recognize. Asa consequence, cellular internalization is slow since it likely occursvia receptor recycling, rather than the more rapid process ofactivation-induced endocytosis.

Only MDA-MB-468 cells took up the aptamer. Uptake was clearly detectedat 22 hr, but increased greatly after 43 hr. To test whether EpCAM-AsiCsare specifically taken up by EpCAM bright cell lines, the 3′ end of theantisense strand of the AsiC was fluorescently labeled. EpCAM+ BPLER, abasal-A TNBC cell line transformed from BPE by transfection with humanTERT, SV40 early region and H-RASV12, took up Alexa-647 EpCAM-AsiCs whenanalyzed after a 24 hr incubation, but BPE cells did not (FIG. 1D).Previous studies have shown that AsiCs are processed within cells byDicer to release the siRNA from the aptamer (10, 12, 15). To verify thatthe released siRNA was taken up by the RNA induced silencing complex(RISC), qRT-PCR was utilized to amplify that PLK1 siRNAimmunoprecipitated with Ago when MDA-MB-468 cells were incubated withPLK1 EpCAM-AsiCs (FIG. 33). No PLK1 siRNA bound to Ago when the samecells were incubated with PLK1 siRNAs.

TNBC cells took up Alexa-467 EpCAM-AsiCs, but no uptake was detectablein BPE cells (FIG. 1E). Next to assess whether gene knockdown wasspecific to EpCAM+ tumors, eGFP knockdown was compared in these samecell lines, which stably express eGFP, by eGFP EpCAM-AsiCs and lipidtransfection of eGFP siRNAs (FIG. 1D). Although transfection of eGFPsiRNAs knocked down gene expression equivalently in BPE and BPLER,Incubation with EpCAM-AsiCs in the absence of any transfection lipidselectively knocked down expression only in BPLER. AsiC knockdown wasuniform and comparable to that achieved with lipid transfection. Next wecompared the specific knockdown of the endogenous AKT1 gene by AKT1AsiCs and transfected AKT1 siRNAs in 6 breast cancer cell lines comparedto normal human fibroblasts (FIG. 1E). AKT1 was selectively knocked downby EpCAM-AsiCs targeting AKT1 only in EpCAM^(bright) luminal and basal-ATNBCs, but not in mesenchymal basal-B TNBCs, fibroblasts or BPE ells(data not shown). As expected, AsiCs targeting eGFP had no effect onAKT1 levels and transfection of AKT1 siRNAs comparably knocked downexpression in all the cell lines studied. Moreover, EpCAM-AsiC knockdownof AKT1 strongly correlated with EpCAM expression (FIG. 1G). Similarresults were obtained when AKT1 protein was analyzed by flow cytometryin stained transfected cells (FIG. 1G, 1H). Thus in vitro knockdown byEpCAM-AsiCs is effective and specific for EpCAM^(bright) tumor cells.

PLK1 EpCAM-AsiCs Selectively Kill EpCAM^(bright) Tumor Cells In Vitro

To explore whether EpCAM-AsiCs could be used as anti-tumor agents inbreast cancer, we examined by CellTiterGlo assay the effect of AsiCsdirected against PLK1, a kinase required for mitosis, on survival of 10breast cancer cell lines that included 5 basal-A TNBCs, 2 luminal celllines, and 3 basal-B TNBCs. EpCAM-AsiCs targeting PLK1, but not controlAsiCs directed against eGFP, decreased cell proliferation in the basal-Aand luminal cell lines, but did not inhibit basal-B cells (FIG. 2A).Lipid transfection of PLK1 siRNAs suppressed the growth of all the celllines. The anti-proliferative effect strongly correlated with EpCAMexpression (FIG. 2B). The reduction in viable EpCAM+ cells afterknockdown was due to induction of apoptosis, assessed by annexinV-propidium iodide staining and caspase activation (data not shown). Todetermine whether ligation of the EpCAM aptamer contributed to theanti-proliferative effect of the EpCAM-AsiC, we compared survival ofcells that were treated with the PLK1 EpCAM-AsiC with cells treated withthe aptamer on its own (FIG. 2C). The aptamer by itself did not have areproducible effect on survival of any breast cancer cell lines,possibly because as a monomeric agent it does not cross-link the EpCAMreceptor to alter EpCAM signaling. Thus the PLK1 EpCAM-AsiC asserts itsspecific anti-tumor effect on EpCAM+ breast cancer cells by geneknockdown.

To determine whether EpCAM-AsiCs specifically target EpCAM+ cells whenmixed with EpCAM^(dim) non-transformed epithelial cells, we incubatedco-cultures of GFP− TNBC cells and GFP+ BPE cells with PLK1 EpCAM-AsiCsor medium and used GFP fluorescence to measure their relative survivalby flow cytometry 3 days later (FIG. 2D, 2E). EpCAM-AsiCs targeting PLK1greatly reduced the proportion of surviving EpCAM+ basal-A tumor cells,but had no effect on survival of an EpCAM− basal-B cell line. Thus PLK1EpCAM-AsiCs are selectively cytotoxic for EpCAM+ tumor cells when mixedwith normal cells.

EpCAM-AsiCs Concentrate in EpCAM+ Breast Tumor Biopsy Specimens

It was next examined whether EpCAM-AsiCs concentrate in human breasttumors relative to normal breast samples within intact tissues. Pairednormal tissue and breast tumor biopsies from 3 breast cancer patientswere cut into cubes with −3 mm edges and placed in Petri dishes. Thetumor sample cells were all EpCAM^(bright) and the normal tissue cellswere EpCAM^(dim) (FIG. 3A). Fluorescently labeled Alexa647-siRNAs (notexpected to be taken up by either normal tissue or tumor),Alexa647-cholesterol-conjugated siRNAs (chol-siRNAs, expected to betaken up by both), or Cy3-EpCAM-AsiCs were added to the culture mediumand the tissues were incubated for 24 hr before harvest. The Cy3 signalof the AsiC, which could be visualized by the naked eye, concentratedonly in the tumor specimens and was not detected in normal tissue (FIG.3B). To quantify RNA uptake, flow cytometry analysis was performed onwashed single cell suspensions of the tissue specimens (representativetumor-normal tissue pair (FIG. 3C), mean±SD of triplicate biospies from3 EpCAM^(bright) paired breast tumor-normal tissue samples (FIG. 3D)).The EpCAM-AsiC was significantly taken up by the tumor, but not normaltissue, while neither took up the unconjugated siRNA and both took upthe chol-siRNA to some extent. Thus, within intact tissue, EpCAM-AsiCsare selectively delivered to EpCAM^(bright) tumors relative to normaltissue.

PLK1 EpCAM-AsiCs Inhibit T-ICs of EpCAM+ Tumors

EpCAM was chosen for targeting in part because EpCAM marks T-ICs andmetastasis-initiating cells (M-IC).^(20,22,26,27,31) To investigatewhether EpCAM-AsiCs inhibit T-ICs, we compared colony and mammosphereformation (T-IC functional surrogate assays) after mock treatment,treatment with paclitaxel or with EpCAM-AsiCs against eGFP or PLK1. PLK1EpCAM-AsiCs more strongly inhibited colony and mammosphere formation ofEpCAM+ basal-A TNBCs and luminal cell lines than paclitaxel, but wereinactive against EpCAM− basal-B TNBCs (FIGS. 4A-C). T-IC inhibition wasspecific, since eGFP AsiCs had no effect. Incubation with PLK1EpCAM-AsiCs, but not eGFP AsiCs, also reduced the proportion of cellswith the phenotype of T-ICs, namely the numbers of CD44+CD24^(low/−) andALDH+ cells specifically in basal-A and luminal breast cancer cell lines(data not shown). To evaluate the effect of EpCAM-AsiCs on tumorinitiation, EpCAM+MB468 cells stably expressing luciferase were treatedovernight with medium or PLK1 or eGFP EpCAM-AsiCs and equal numbers ofviable cells were then implanted sc in nude mice. PLK1 EpCAM-AsiCscompletely blocked tumor formation assessed by in vivo tumor cellluminescence (data not shown). In contrast similar treatment of basal-BMB436 cells had no effect on tumor initiation (data not shown). ThusPLK1 EpCAM-AsiCs inhibit in vitro T-IC assays and tumor initiationselectively for EpCAM+ breast cancers.

Subcutaneously Administered EpCAM-AsiCs are Selectively Taken Up byDistant EpCAM+ TNBCs

To be clinically useful, EpCAM-AsiCs need to be taken up by disseminatedtumor cells. Intravenous injection of fluorescent EpCAM-AsiCs in thetail vein of mice did not lead to significant AsiC accumulation withinsubcutaneous tumors implanted in the flanks of nude mice (data notshown), probably because their size (˜25 kDa) is below the threshold forkidney filtration and they are rapidly excreted. Linkage to polyethyleneglycol greatly enhanced the circulating half-life, tumor accumulationand antitumor therapeutic effect of PSMA-AsiCs in a mouse xenograftmodel of prostate cancer.¹¹ However, to see if this modification couldbe bypassed, we examined by live animal epifluorescence imaging whethersc injection of Alexa750-labeled eGFP EpCAM-AsiCs in the scruff of theneck of 7 mice led to accumulation in distant EpCAM+MB468 and EpCAM−MB231 TNBCs implanted sc in each flank (FIG. 5A, 5B). Within a day ofinjection, EpCAM-AsiCs concentrated only in the EpCAM+ tumor andpersisted there for at least 4 days. The EpCAM-AsiCs were detectedaround the injection site on day 2, but were only found within theEpCAM+ tumor on day 4.

PLK1 EpCAM AsiCs Cause Regression of Basal-A TNBC and Her2 Breast CancerXenografts

Because sc injected EpCAM-AsiCs concentrated in distant EpCAM+ tumors,we next looked at whether sc injection of PLK1 EpCAM-AsiCs couldselectively inhibit the growth of an EpCAM+ TNBC xenografted tumor.EpCAM+MB468-luc cells were implanted in Matrigel in one flank of a nudemouse and EpCAM− MB231-luc-mCherry cells were implanted on the oppositeflank. Once the luciferase signal of both tumors was clearly detectedabove background, groups of 5-6 mice were mock treated or injected scwith 5 mg/kg of EpCAM-AsiCs targeting PLK1 or eGFP every 3 d for 2 wks.Tumor growth was followed by luminescence. All the EpCAM+ tumors rapidlycompletely regressed only in mice that received the PLK1-targeting AsiCs(FIG. 6A, 6B). The EpCAM+ tumors in mice treated with eGFP-targetingAsiCs and all the EpCAM− tumors continued to grow. This experiment wasrepeated with similar results after injection of PLK1 AsiCs. Tumors alsocontinued to grow without significant change in additional groups ofcontrol mice treated with just the EpCAM aptamer or the PLK1 siRNA (datanot shown) and into mice bearing Her2+ MCF10A-CA1a (FIG. 34). Thus scinjected PLK1 EpCAM-AsiCs show specific antitumor activity againstbasal-A TNBCs and EpCAM+ human xenografts.

Discussion

Targeted therapy so far has relied on using tumor-specific antibodies orinhibitors to oncogenic kinases. No one before has shown that anunconjugated AsiC can have potent antitumor effects or that AsiCs couldbe administered sc. There is currently no targeted therapy for TNBC orfor T-ICs. Developing targeted therapy for TNBC and developing ways ofeliminating T-ICs are important unmet goals of cancer research.

It is demonstrated herein that EpCAM-AsiCs can be used to knockdowngenes selectively in epithelial breast cancer cells and their stemcells, sparing normal epithelial cells and stroma, to cause tumorregression and suppress tumor initiation. In one very aggressive TNBCxenograft model, the EpCAM-AsiCs caused complete tumor regression afteronly 3 injections. This is a flexible platform for targeted therapy,potentially for all the common epithelial cancers, which uniformlyexpress high levels of EpCAM.

Although EpCAM-AsiCs targeting PLK1 was used herein, the siRNA can bevaried to knockdown any tumor dependency gene that would be customizedto the tumor subtype or the molecular characteristics of an individualpatient's tumor. AsiC cocktails targeting more than one gene would beideal for cancer therapeutics to lessen the chances of developing drugresistance. Targeted cancer therapy so far has relied on usingtumor-specific antibodies or small molecule inhibitors to oncogenickinases. Using EpCAM as an AsiC ligand and developing RNAi therapy totarget cancer stem cells is novel. No one before has shown that anunconjugated AsiC can have potent antitumor effects or that AsiCs couldbe administered sc. Moreover, preliminary studies of sc administeredCD4-AsiCs in humanized mice showed strong knockdown in CD4 cells in thespleen and distant lymph nodes, suggesting that AsiCs targetingreceptors on cells located elsewhere in the body could also beadministered sc. There is currently no targeted therapy for TNBC or forT-ICs. Targeted delivery has the advantage of reduced dosing and reducedtoxicity to bystander cells.

The major obstacle to harnessing RNAi for cancer is delivering smallRNAs into disseminated cells. Described herein is the use of AsiCs toovercome this obstacle. Described herein is a new class of potentanticancer drugs. AsiCs are a flexible platform that can targetdifferent cell surface receptors and knockdown any gene or combinationof genes. {Burnett, 2012 #18447; Zhou, 2011 #18448; Thiel, 2010 #18445}By changing the aptamer, the AsiC platform can tackle the deliveryroadblock that has thwarted the application of RNAi-based therapy tomost diseases. This approach is ideal for personalized cancer therapy,since the choice of genes to target can be adjusted depending on atumor's molecular characteristics. Moreover RNA cocktails can knockdownmultiple genes at once to anticipate and overcome drug resistance. AsiCsare the most attractive method for gene knockdown outside the liver.They are better than complicated liposomal, nanoparticle or conjugatedmethods of delivering RNAs because they are a single chemical entitythat is stable in the blood, easy to manufacture, nonimmunogenic, ableto readily penetrate tissues and are not trapped in the filteringorgans.

An important cancer research goal is to eliminate T-ICs (cancer stemcells). T-ICs are relatively resistant to chemotherapy and are thoughtresponsible for tumor relapse and metastasis. {Federici, 2011 #19371}The AsiCs described herein target (epithelial) T-ICs with highefficiency. As such they may eliminate this aggressive subpopulationwithin tumors at risk for progressive disease (see FIG. 6A, 6B).

The small size of the EpCAM aptamer used here is ideal for an AsiC drug,since RNAs <60 nt can be efficiently synthesized.

In addition to their potential therapeutic use, EpCAM-AsiCs are also apowerful in vivo research tool for identifying the dependency genes oftumors and T-ICs to define novel drug targets. In principle, aptamerchimeras could be designed to deliver not only siRNAs but also miRNAmimics or antagomirs, antisense oligonucleotides that function by othermechanisms besides RNAi, or even longer mRNAs or noncoding RNAs (50,51). They could also be designed to incorporate more than one aptamer,multiple siRNAs, or even toxins or small molecule anticancer drugs.

Its small size is ideal for an AsiC drug, since RNAs <60 nt can beefficiently synthesized. Not only is the siRNA targeted to the tumor,but the drug targets can also be chosen to attack the tumor's Achilles'heels by knocking down tumor dependency genes. This flexibility can beused for personalized cancer therapy that targets the molecularvulnerabilities of an individual patient's cancer.

Material and Methods

Cell culture. Human BPE and BPLER cells were grown in WIT medium(Stemgent). MB468 were transduced with a luciferase reporter. All otherhuman cell lines were obtained from ATCC and grown in MEM (MCF7, BT474),McCoy's 5A (SKBR3), RPMI1640 (HCC1806, HCC1143, HCC1937, HCC1954,HCC1187, MB468, T47D) or DMEM (MB231, BT549, MB436) all supplementedwith 10% FBS, 1 mM L-glutamine and penicillin/streptomycin (Gibco)unless otherwise indicated. 4T1 mouse breast cancer cells were grown in10% FBS DMEM. For in vivo imaging, MB468 cells stably expressing Fireflyluciferase (MB468-luc) were used and MB231 cells stably expressingFirefly luciferase and mCherry (MB231-luc-mCherry) were selected afterinfection with pLV-Fluc-mCherry-Puro lentivirus (provided by AndrewKung, Columbia University). MB231 Cells were selected with puromycin.

For uptake and silencing treatment, cells were plated at low density(10,000 cells/well in 96-well plates) and treated immediately. All AsiCand siRNA treatments were performed in either OptiMEM or WIT medium.Cell viability was assessed by CellTiter-Glo (Promega) or by Trypan-Bluestaining in 96-well plates.

For colony formation assay, 1,000 viable cells were treated for 6 h inround bottom 96-well plates and then transferred to 10-cm plates inserum-containing medium. Medium was replaced every 3 d. After 8-14 d,cells were fixed in methanol (−20 C) and stained with crystal violet.For sphere formation assay, 1,000/ml viable cells were treated for 6 hin round bottom 96-well plates and then cultured in suspension inserum-free DMEM/F12 1:1 (Invitrogen), supplemented with EGF (20 ng/ml,BD Biosciences), B27 (1:50, Invitrogen), 0.4% bovine serum albumin(Sigma) and 4 μg/ml insulin (Sigma). Spheres were counted after 1 or 2weeks.

siRNA Transfection.

Cells were transfected with Dharmafect I per the manufacturer'sprotocol. See FIG. 9 for all siRNA sequences.

Flow Cytometry.

For flow cytometry, cells were stained as previously described (Yu, F.et al (2007). let-7 Regulates Self Renewal and Tumorigenicity of BreastCancer Cells. Cell 131, 1109-1123. ), briefly, direct immunostaining ofEpCAM and AKT1 was performed using 1:50 dilutions of hAb for 30-60minutes at 4° C. (BioLegend/BD). Cells were stained in PBS containing0.5% FCS, 1 mM EDTA, and 25 mM HEPES. Samples were washed twice in thesame buffer. Data was acquired using FACS-Canto II (BD Biosciences).Analyses were performed in triplicate and 10,000 gated events/samplewere counted. All data analysis was performed using FlowJo (TreestarInc.).

RNA Analysis.

qRT-PCR analysis was performed as described (Petrocca, F., et al.(2008). E2F1-regulated microRNAs impair TGFbeta-dependent cell-cyclearrest and apoptosis in gastric cancer. Cancer Cell 13, 272-286).Briefly, total RNA was extracted with Trizol (Invitrogen) and cDNAprepared from 1000 ng total RNA using Thermoscript RT kit (Invitrogen)as per the manufacturer's SYBR Green Master Mix (Applied Biosystems) anda BioRad C1000 Thermal Cycler (Biorad). Relative CT values werenormalized to GAPDH and converted to a linear scale.

Collagenase Digestion of Human Breast Tissue.

Fresh breast or colon cancer and control biopsies were received from theUMASS Tissue Bank, samples were cut into 3×3×3 mm samples and placed ina 96 well plate with 100 ul RPMI. Samples were treated with eitherAlexa647-siRNA-GFP, Alexa647-chol-siRNA-GFP or Cy3-AsiC-GFP for 24 hr.Samples were photographed and digested. Three samples from eachtreatment were pooled and put in 10 ml RPMI containing 1 mg/mlcollagenase II (Sigma-Aldrich) for 30 minutes at 37° C. with shaking.Samples were disrupted in a gentleMACS dissociator (Miltenyi) using thespleen program for 30 minutes at 37° C. both before and aftercollagenase digestion. Cell suspensions were passed through a 70-μm cellstrainer (BD Falcon), washed with 30 ml RPMI, and stained for flowcytometry.

Animal Experiments.

All animal procedures were performed with Harvard Medical School andBoston Children's Hospital Animal Care and Use Committee approval. Nudemice were purchased from the Jackson Laboratory.

In Vivo Experiments.

For tumor initiation studies 8-week old female Nu/J mice (Stock #002019,Jackson Laboratories) were injected subcutaneously with MB468-luc(5×10⁶) cells pretreated for 24 h with EpCAM-AsiC-GFP, EpCAM-AsiC-PLK1or untreated. Cells were trypsinized with Tryple Express (Invitrogen),resuspended in WIT media and injected subcutaneously in the flank.Following intraperitoneal injection of 150 mg/kg D-luciferin (CaliperLife Sciences) luminescent images of the whole body were taken every 5days for a total of 20 days using the IVIS Spectra system (Caliper LifeSciences).

For AsiC uptake experiments MB468-luc (5×10⁶) and MB231-luc-mCherry(5×10⁵) cells trypsinized with Tryple Express (Invitrogen), wereresuspended in a 1:1 WIT-Matrigel solution and injected subcutaneouslyin the flank of 8-week old female Nu/J mice (Stock #002019, JacksonLaboratories). Tumors size was analyzed daily using the IVIS Spectrasystem (Caliper Life Sciences). After 5 days tumors were clearly visibleand mice were injected subcutaneously in the neck area withAlexa750-EpCAM-AsiC-GFP (0.5 mg/kg). Localization of the AsiC comparedto the tumor was tested every 48 h for 7 days.

For tumor inhibition studies, MB468-luc (5×10⁶) and MB231-luc-mCherry(5×10⁵) cells trypsinized with Tryple Express (Invitrogen), resuspendedin a 1:1 WIT-Matrigel solution and injected subcutaneously in the flankof 8-week old female Nu/J mice (Stock #002019, Jackson Laboratories).Tumors size was analyzed daily using the IVIS Spectra, after 5 daystumors were clearly visible. Mice bearing tumors of comparable size wererandomized into 5 groups and treated with 5 mg/kg of EpCAM-AsiC-PLK1,EpCAM-AsiC-GFP, EpCAM-Aptamer, siRNA-PLK1 or untreated. Mice weretreated every 72 h for 14 days.

All Images were analyzed using Living Image® software (Caliper LifeSciences).

Statistical Analysis.

Student's t-tests, computed using Microsoft Excel, were used to analyzethe significance between the treated samples and the controls where thetest type was set to one-tail distribution and two-sample equalvariance. To assess innate immune stimulation, one-way analysis ofvariance (ANOVA) with Bonferroni's Multiple comparison test wasperformed using GraphPad Prizm 4 software (GraphPad Software, San Diego,Calif.). P<0.05 was considered significant.

Measurement of Innate Immune Stimulation.

Mice were injected sc with eGFP EpCAM-AsiCs (5 mg/kg) or ip withPoly(I:C) (5 or 50 mg/kg). Serum samples, collected at baseline and 6and 16 hr after treatment were stored at −80° C. before measuring IFNβ,IL-6 and IP-10 using the ProcartaPlex Multiplex Immunoassay(Affymetrix/eBioscience, San Diego, Calif.). Spleens, harvested atsacrifice 16 hr post treatment, were stored in RNAlater (Qiagen) beforeextracting RNA using TRIZOL (Invitrogen) with the gentleMACS Dissociator(MACS Miltenyi Biotec, San Diego, Calif.). cDNA was synthesized usingSuperscript III and random hexamers (Invitrogen) and PCR was performedusing SsoFast EvaGreen Supermix and a Bio-Rad CFX96 Real-Time PCR System(Bio-Rad Laboratories, Hercules, Calif.) using the following primers:

Gapdh forward: (SEQ ID NO: 4) 5′-TTCACCACCATGGAGAAGGC-3′, Gapdh reverse:(SEQ ID NO: 5) 5′-GGCATGGACTGTGGTCATGA-3′, ifnb forward: (SEQ ID NO: 6)5′-CTGGAGCAGCTGAATGGAAAG-3′, ifnb reverse: (SEQ ID NO: 7)5′-CTTGAAGTCCGCCCTGTAGGT-3′, il-6 forward: (SEQ ID NO: 8)5′-TGCCTTCATTTATCCCTTGAA-3′, il-6 reverse: (SEQ ID NO: 9)5′-TTACTACATTCAGCCAAAAAGCAC-3′, ip-10 forward: (SEQ ID NO: 10)5′-GCTGCCGTCA1TTTCTGC-3′, ip-10 reverse: (SEQ ID NO: 11)5′-TCTCACTGGCCCGTCATC-3′, oas-1 forward: (SEQ ID NO: 12)5′-GGAGGTTGCAGTGCCAACGAAG-3′, oas-1 reverse: (SEQ ID NO: 13)5′-TGGAAGGGAGGCAGGGCATAAC-3′, stat1 forward: (SEQ ID NO: 14)5′-TTTGCCCAGACTCGAGCTCCTG-3′, stat1 reverse: (SEQ ID NO: 15)5′-GGGTGCAGGTTCGGGATTCAAC-3′.

EpCAM PLK1 sense GCG ACU GGU UAC CCG GUC GUU UUG SEQ ID NO: 1AAG AAG AUC ACC CUC CUU AdTdT EpCAM PLK1 anti-UAA GGA GGG UGA UCU UCU UCA dTdT SEQ ID NO: 2 sense EpCAM PLK1 anti-GCG ACU GGU UAC CCG GUC GUU UUAA SEQ ID NO: 3 senseGGA GGG UGA UCU UCU UCA dTdT EpCAM aptamer GCG ACU GGU UAC CCG GUC GUU USEQ ID NO: 33

EpCAM is over expressed in basal A and luminal but not basal B breastcancer cell lines (data not shown). FACS was performed with 8 differentbreast cancer cell lines, testing EpCAM expression levels by flowcytometery using a hEpCAM Antibody. EpCAM is over expressed in all basalA and luminal cells lines and not in basal B.

Specific decrease in cell viability in Basal A breast cancer cell linesis PLK1 dependent. Ten different breast cancer cell lines representingbasal A, B and luminal cells were treated with either EpCAM-AsiCtargeting PLK1 or just the EpCAM-aptamer and compared to untreatedcontrols. None of the cell lines treated with EpCAM-aptamer displayeddecrease in cell viability, while basal A and luminal cell linesdisplayed a decrease in cell viability following treatment withEpCAM-AsiC targeting PLK1 (data not shown).

EpCAM-AsiC is taken up by both healthy and colon cancer biopsies.Cy3-EpCAM-AsiC targeting GFP, Alexa647-siRNA-GFP orAlexa647-chol-siRNA-GFP (2 μM of each) were added to colon cancer andcontrol explants and incubated for 24 h before tissues were digestedwith collagenase to a single cell suspension and analyzed by flowcytometry. EpCAM-AsiC, siRNA and chol-siRNA penetrated both tumor andhealthy tissue with similar efficacy. At day 5 the tumors were removedand visualized to validate that the Alexa750 labeled EpCAM-AsiCtargeting GFP indeed entered the tumors. Increased level of Alexa750 isnegatively correlated with mCherry levels (n=8, *P<0.05, t-test EpCAM+versus EpCAM− cells) (data not shown).

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Aptamermediated delivery of splice-switching    oligonucleotides to the nuclei of cancer cells. Nucleic Acid Ther    2012; 22:187-195.-   51. Esposito C L, Cerchia L, Catuogno S, De Vita G, Dassie J P,    Santamaria G et al. Multifunctional aptamer-miRNA conjugates for    targeted cancer therapy. Mol Ther 2014; 22:1151-1163.

Example 2

Described herein is the development of targeted siRNA delivery(aptamer-siRNA chimeras (AsiC)) that use chimeric RNAs composed of astructured RNA, called an aptamer, selected for high affinity binding toa cell surface protein, that is covalently linked to an siRNA. TheseAsiCs are taken up by cells expressing a receptor that the aptamerrecognizes and are processed within cells to release the active siRNA.This is a flexible platform that can be modified to target differentcells by targeting specific cell surface receptors and can be designedto knockdown any gene or combination of genes.

The aptamer, was selected for high affinity binding to human EpCAM(CD326 or ESA) which is expressed on all epithelial cells, but is muchmore highly expressed on epithelial cancers including poorlydifferentiated breast cancers, such as basal-like TNBC. All the commoncancers (lung, pancrease, prostate, breast and colon) have high EpCAMexpression and can potentially be targeted.

It is demonstrated herein that epithelial breast cancer cells, but notmesenchymal or normal epithelial cells, selectively take up EpCAM-AsiCsand undergo gene knockdown in vitro. Moreover, the extent of knockdownstrongly correlates with EpCAM levels. Knockdown of PLK1, a gene neededfor mitosis, using EpCAM-AsiCs eliminates cancer cell line growth andstem cell properties including colony and mammosphere formation andtumor initiation in xenografts. This platform can be used to eliminatecancer cells and the malignant cancer stem cells within epithelialtumors.

EpCAM AsiCs can be delivered specifically to basal-like tumors andinhibit tumor growth. These AsiCs can also be a powerful research toolfor identifying the genes that T-IC cells depend on, which could be goodtargets for either conventional drugs or RNAi-based drugs.

Example 3

A ubiquitous mechanism for regulating gene expression is called RNAinterference. It uses small RNAs bearing a short complementary sequenceto block the translation of genetic information into proteins.Harnessing this endogenous process offers the exciting possibility totreat disease by knocking down expression of disease-causing genes. Themajor obstacle is delivering small RNAs into cells, where the RNAinterference machinery lies. In the past year, preliminary clinicalstudies have shown very promising results without significant toxicityin a few diseases caused by aberrant gene expression in the liver.However, delivery to the liver, an organ that traps particles in theblood, is easier to accomplish than delivering drugs to metastatic tumorcells. Described herein is a strategy for targeting RNAs into epithelialcancer cells that is especially good at targeting the most aggressivetype of breast cancer, triple negative breast cancer (TNBC). Moreover,it also targets the most malignant subpopulation in most breast cancers,which are called cancer stem cells. These cells are resistant tochemotherapy drugs and are thought responsible for tumor recurrence andmetastasis. An important goal of current cancer research is to replacecytotoxic chemotherapy drugs that are toxic for both cancer cells andnormally dividing cells (such as the blood forming cells and cellslining the gut) with agents that have selective activity against thetumor, especially against the cancer stem cells within the tumor.

Targeted therapy for one type of breast cancer (Her2+) hasrevolutionized treatment and significantly improved survival. There iscurrently no targeted therapy for TNBC or for breast cancer stem cells.

Described herein in are data demonstrating that RNAs that link aninterfering RNA to a structured RNA (aptamer) that recognizes a cellsurface protein can knockdown gene expression in aggressive breastcancer cells. Aptamers that bind to proteins highly expressed on breastcancer stem cells and most TNBC cells can knock down proteins requiredfor cancer cell division or survival specifically in the most commonsubtype of TNBC. These RNAs can be tested, e.g., in both tissue cultureand in mouse models of TNBC. Described herein is a platform forharnessing RNA-based drugs to treat poor prognosis breast cancer anddemonstration in a mouse model of its efficacy.

Ultimate applicability for treating breast cancer (which patients, howwill it help them, clinical applications/benefits/risks, projected timeto patient-related outcome) The proteins that this therapy can targetare expressed on all epithelial cancer cells, but are more stronglyexpressed on the least differentiated, and hence most malignant, cancercells. This approach could be used to treat not only most epithelialbreast cancers (and most breast cancer cells are epithelial), but alsohas the potential to treat the common cancers, including colon, lung,pancreas, and prostate. Our focus is on the most aggressive and poorestprognosis breast cancer, TNBC, which preferentially strikes down youngwomen and women from minority populations. This approach permits a newplatform for breast cancer therapy. Any cancer-causing or promotinggene, or combinations of genes, could be knocked down, making thisstrategy ideal for the coming era of personalized cancer therapy inwhich each patient's therapy will be customized according to themolecular characteristics of her individual tumor.

Moreover, if a tumor is nonresponsive or becomes resistant, the cocktailof target genes could be nimbly adjusted. Because normal epithelialcells express low levels of the proteins used for targeting, there maybe some uptake and toxicity to normal epithelial cells, which isevaluated herein. However, the platform is flexible so that thetherapeutic siRNA cargo can be chosen to kill tumor cells with minimaltoxicity to normal cells.

Described herein are the design and testing in mouse TNBC models ofseveral molecules capable of causing tumor-specific gene knockdown andtumor suppression.

There is no targeted therapy for TNBC or for highly malignanttumor-initiating cell subpopulations within breast cancers.

Triple negative breast cancer (TNBC) has the worst breast cancerprognosis. 1-4 There is no targeted therapy, and TNBCs often relapse.Described herein is the development of small RNA-based drugs thatknockdown tumor dependency genes in basal-like (or basal-A) TNBCs. Inprinciple RNA interference (RNAi) can be harnessed to knockdowndisease-causing genes to treat any disease. 5-9 However, convertingsmall RNAs into drugs is challenging. Recent Phase I and II clinicaltrials have shown dramatic and durable gene knockdown in the liver(˜80-95%, lasting for almost a month after a single injection) with nosignificant toxicity. 10-16 Realizing the potential of gene knockdownfor treating cancer, however, requires a robust method to deliver RNAsinto disseminated cancer cells, which the liver-targeting RNAs areunable to do. 7 An ideal therapy would selectively knockdown genes incancer cells, sparing normal cells to minimize toxicity. 17

AsiCs are composed of an RNA aptamer (a structured RNA with highaffinity for a receptor)18,19 covalently linked to an siRNA (FIGS.10A-10B).

Described herein is the use of an AsiC to knockdown genes in epithelialcancers using an EpCAM aptamer. 37 EpCAM, the first described tumorantigen, is highly expressed on all common epithelial cancers. 38-45 Onepithelial breast cancers, EpCAM is ˜400-fold more abundant than onnormal breast tissue. 46 EpCAM39,45,47-53 is also highly expressed onmost epithelial cancer tumor-initiating cells (T-IC, also known ascancer stem cells). 39,45,47-53

The EpCAM aptamer has high affinity (12 nM) and is short (19 nt), whichis ideal for an AsiC drug, since RNAs <60 nt can be cheaply andefficiently synthesized. The EpCAM-AsiCs consist of a long 42-44 ntstrand (19 nt aptamer+3 nt linker+20-22 nt siRNA sense strand) annealedto a 20-22 nt antisense (active) siRNA strand (FIG. 10B). They arecommercially synthesized with 2′-fluoropyrimidines, which enhance serumstability (T1/2>3d) and block innate immune recognition. 28,54-56

EpCAM targeting can cause selective gene knockdown in basal-like TNBCs,relative to normal epithelia. Selective knockdown will reduce both thedrug dose and normal tissue toxicity. In normal epithelia, EpCAM is onlyexpressed on basolateral gap junctions, where it may not be accessible.In epithelial cancers, it's both more abundant and distributed along thewhole cell membrane. EpCAM promotes adhesion, and also enhancesproliferation and invasiveness. Proteolytic cleavage of EpCAM releasesan intracellular fragment that increases transcription of stem cellfactors. The oncogenic properties of EpCAM may make it difficult fortumor cells to develop resistance by down-modulating EpCAM. The numberof EpCAM+ circulating cells is linked to poor prognosis in breastcancer. In fact, enumerating circulating EpCAM+ cells is the basis of anFDA-approved method for monitoring metastatic breast, colon and prostatecancer treatment. In our studies, 9 of 9 basal-A TNBC and luminal breastcancer cell lines were strongly EpCAM+, while a normal breast cancerepithelial line and mesenchymal TNBCs had close to background levels(FIG. 1B). Thus most basal-like TNBCs and luminal breast cancers willlikely be targeted by EpCAM-AsiCs. In preliminary data, EpCAM-AsiCsselectively knocked down expression in EpCAM+ breast and colon cancercell lines but not in normal epithelial cells or mesenchymal tumorcells; knockdown was uniform and comparable to lipid transfection, butlipid transfection uniformly knocked down gene expression in all thelines. (FIG. 3A-3C)

AKT1 knockdown and inhibition of cell proliferation by EpCAM-AsiCsagainst PLK1, a kinase required for mitosis, correlated with EpCAMlevels. When normal transformed epithelial cells (BPE) 57 were mixedwith epithelial TNBC cell lines, EpCAM− AsiCs caused PLK1-sensitive celldeath only in the tumor cells, sparing BPE cells (not shown). Moreoverwhen tumor biopsies and normal tissue biopsies were coincubated withfluorescent AsiCs, only the tumors took up the AsiCs and fluoresced (notshown). These results suggest that EpCAM-AsiCs are specific forepithelial tumor cells compared to normal epithelia.

EpCAM also marks T-ICs. 40,45,58 An important goal of cancer research isto develop a way to target T-ICs. Although the stem cell hypothesis iscontroversial and may not apply to all cancers, there is good evidencethat breast cancers contain a T-IC subpopulation. 59-82 T-Ics arerelatively resistant to chemotherapy and are also thought responsiblefor tumor relapse and metastasis. The AsiCs described herein aredesigned to target (epithelial) T-ICs with high efficiency. As such theymay be suitable for eliminating this aggressive subpopulation withinpatients at risk for relapse. To investigate whether EpCAM-AsiCs inhibitTNBC T-ICs, we compared mammosphere and colony formation (in vitrosurrogates of T-IC function) of breast tumor cells that were mocktreatedor treated with EpCAM-AsiCs against eGFP or PLK1. PLK1 EpCAM-AsiCs, butnot control GFP AsiCs, eliminated mammosphere and colony formation ofbreast luminal and basal-like TNBC cell lines (FIG. 11D). PLK1EpCAM-AsiCs also reduced CD44+CD24low and Aldefluor+ cells (not shown).Importantly, treatment with PLK1 EpCAM-AsiCs eliminated tumor initiationby basal-like TNBCs, but, as expected, had no effect on basal-B TNBCtumor initiation (data not shown). Luciferase-expressing cell lines weremock-treated or treated overnight with AsiCs before orthotopicimplantation in the mammary fatpad.

AsiCs targeting EphA2, important in EGF receptor signaling. 83-92 arealso contemplated herein. EphA2 is expressed on epithelial andmesenchymal (basal-A and basal-B, respectively) TNBC cell lines,including their T-ICs, but less than EpCAM and only weakly on otherbreast cancers. Inhibiting EphA2 reduces tumor growth and angiogenesisin multiple cancer models. Furthermore, EphA2 is selectively accessibleon cancer cells, but not normal cells.

Also contemplated herein are mouse-human cross-reactive AsiCs, whichwill be valuable for future drug development, since they will enable usto evaluate toxicity and effectiveness in spontaneous mouse tumormodels.

AsiCs targeting EphA2 can produce dual functioning RNAs that bothinhibit EphA2 signaling and cell proliferation and knockdown genes.

AsiCs are ideal for personalized cancer therapy, since the genestargeted for knockdown can be adjusted to the molecular characteristicsof a tumor. Moreover cocktails of RNAs can be assembled to knockdownmultiple genes at once for combinatorial therapy to anticipate andovercome drug resistance. AsiCs not only target the drug to the tumor,but the siRNAs can also be chosen to attack the specific Achilles' heelsof the tumor. siRNAs also provide a unique opportunity to target“undruggable” genes. AsiCs that knock down tumor dependency genes,required for tumor, but not normal cell, survival, should have reducedtoxicity. To identify genetic dependencies of basal-like TNBCs that wecould knockdown, we performed a genomewide siRNA lethality screencomparing 2 TNBC cell lines—basal-like BPLER and myoepithelial HMLERcells, human 10 breast epithelial cells transformed with the sameoncogenes in different media. 57,93

Although essentially isogenic, BPLER are highly malignant and enrichedfor T-ICs, forming tumors in nude mice with only 50 cells, while HMLERrequire >105 cells to initiate tumors. The screen identified 154 geneson which BPLER, but not HMLER, depended. Proteasome genes were highlyenriched (P<10-14). BPLER dependency gene expression correlated withpoor prognosis in breast, but not lung or colon, cancer. Because TNBCsare heterogeneous 1,3,4,94, to identify shared dependencies inbasal-like TNBCs, we did another screen to test 17 breast cancer celllines for their dependency on the 154 BPLER dependency genes(unpublished). Although many of the BPLER dependencies were shared withonly a subset of basal-like TNBC cell lines, the proteasome, MCL1, somesplicing genes, and a few other novel genes stood out because virtuallyall (at least 8 of 9) basal-like TNBC lines were dependent on thesegenes, but normal cells were not. As the screen predicted, theproteasome inhibitor bortezomib both killed basal-A TNBCs and alsoblocked T-IC function, assessed by colony and mammosphere formation,again mostly selectively in basal-like TNBCs. Brief exposure tobortezomib also inhibited colony formation and tumor inhibition of amouse epithelial TNBC line. Bortezomib strongly inhibited tumor growthof multiple human basal-A lines and primary TNBCs that arosespontaneously in Tp53+/− mice, but not basal-B or luminal cell lines.Bortezomib also blocked metastatic lung colonization of IV-injected TNBCcells. However, bortezomib does not penetrate well into solid tumors.The maximum tolerated dose was needed to inhibit proteasome activity andsuppress tumors. Although tumor penetration may improve with proteasomeinhibitors in development, proteasome gene knockdown might provide moresustained and efficient proteasome inhibition.

EpCAM− and EphA2-AsiCs can be used for targeted gene knockdown to treatbasal-like TNBC cancers, sparing normal cells, and eliminate the T-Icswithin them. There may be some uptake in normal epithelial cells thatweakly express EpCAM or EphA2, but gene knockdown will be concentratedin aptamer ligandbright tumor cells.

It can be determined which breast cancer subtypes EpCAM− and EphA2-AsiCstarget and determine how aptamer ligand level affects gene silencing.uptake/knockdown in cancer tissues vs normal epithelium can also beevaluated. EpCAM-AsiCs can be compared with EphA2-AsiCs foreffectiveness in causing knockdown in basal-like TNBCs. It can bedetermined whether EpCAM-AsiCs and EphA2-AsiCs can target T-ICs toinhibit tumor initiation.

Pharmacokinetics (PK)/pharmacodynamics (PD) studies of EpCAM− andEphA2-AsiCs can be performed using live animal imaging of orthotopicTNBC xenografted mice. Treated tissue samples and animals can beexamined for toxicity and innate immune activation, and AsiCs will bechemically modified if needed to improve PK/PD or reduce toxicity. Asproof of principle, the antitumor effect of knockdown of PLK1 will beassessed. Suppression of recently identified basal-A TNBC dependencygenes, such as MCL1 and proteasome genes can be accomplished accordingto the methods described herein.

Contemplated herein are:

-   -   cross-species reactive aptamers that recognize EpCAM and EphA2        and are internalized selectively into basal-A TNBCs vs normal        epithelial cells    -   verification of selective uptake, gene silencing and cytotoxic        effect in vitro of TNBC-targeting AsiCs in breast cancer cell        lines vs normal epithelial cells, determination of the subtypes        of breast cancer cell lines they transfect and evaluation of        their potential to transfect and eliminate breast T-Ics    -   Evaluation of systemic delivery and tumor concentration in vivo,        definition of PK and PD and maximally tolerated dose of        TNBC-targeting AsiCs, and evaluation of the antitumor effect of        optimized TNBC-targeting AsiCs that knockdown PLK1 and        dependency genes of basal-like TNBC in human TNBC cell line        models of primary and metastatic cancer in mice

Selection of TNBC-targeting aptamers. Aptamers that bind to a chosentarget are identified by iterative screening of combinatorial nucleicacid sequence libraries of vast complexity (typically 1012-1014 distinctsequences) by a process termed SELEX (Systematic Evolution of Ligands byExponential enrichment). 95,96 In the classic method, the RNA library isincubated with the protein target and the RNAs that bind are separatedand amplified to generate a pool of binding RNAs. These are againapplied in multiple cycles to generate increasingly enriched highaffinity RNA pools. Identification of the sequences that emerge aftermultiple rounds of SELEX was previously accomplished by cloning andsequencing <100 individual sequences.

While this often provided a sufficient number of winning sequences toidentify aptamers, the number of sequences that were analyzed was quitesmall in comparison with the sequence complexity of evolvedoligonucleotide pools. With many selection cycles, some effectiveaptamer sequences that are not efficiently amplified may be depleted andlost. Next generation deep sequencing methods and bioinformatics canpermit evaluation of more sequences within early cycle SELEX sequencepools to identify winning aptamer sequences at earlier selection rounds,thus reducing the time and resources needed to complete identificationof high affinity aptamers. 30,97-104

An important property of aptamers useful for incorporation into AsiCs isefficient internalization into cells. Some ligands of cell-surfaceproteins are efficiently internalized after binding their cell surfaceprotein targets, while others are not. Another strategy (“toggle SELEX”)selects for cross-reactive aptamers that recognize the same ligand fromdifferent species, a useful attribute for preclinical development. Bytoggling cycles between selection with orthologous protein ligands(e.g., mouse and human forms), it is possible to enrich forcross-species reactive aptamers. 105

These SELEX techniques can permit identification of high affinitycross-species reactive aptamers for EpCAM and EphA2 that areinternalized into human (and mouse) basal-like TNBCs, but not into anormal immortalized epithelial cell line. To select additional EpCAM andEphA2 aptamers that have antagonistic activity and/or cross-recognizethe corresponding mouse antigen (the published EpCAM aptamer does notrecognize mouse EpCAM (data not shown)), we can toggle betweencommercially available mouse and human purified, recombinant targetproteins, starting with a library of 1012 RNA sequences containing2′-fluoropyrimidines. This library of 51 nt long oligonucleotides isdesigned with a random region of 20 nucleotides flanked by constantregions of known sequence for PCR amplification at each selection round.Previously described methods will be used to select for high affinityRNAs that bind to immobilized C-terminal tagged proteins. 37 (Thisleaves the N-terminal region exposed to facilitate selection of aptamersthat recognize the extracellular domain.) A tagged control protein canbe used to pre-clear the RNA aptamer library to remove non-specificbinders. 7-10 iterative rounds of SELEX can be performed to enrich forspecific aptamers. Enrichment after each round can be monitored bySurface Plasmon Resonance. Enriched pools that show specific binding canbe sequenced using high-throughput sequencing. Sequences can be chosenfor experimental validation using bioinformatics analysis of theenriched library sequences as described. 97,98,106

The top 10-15 sequences from the high throughput sequencing andbioinformatics analysis can be evaluated by Surface Plasmon Resonance toassess relative binding affinities as described, 99,106 using thepreviously characterized human aptamers for comparison.

An alternative approach to dentify high affinity cross-reactiveaptamers, is cellinternalization SELEX, positively selecting on 293Tcells transfected to expression human or mouse EpCAM and preclearing oncells expressing a control protein. The ability of the 5 highestaffinity aptamers to be internalized into EpCAM/EphA2+ cells will becompared to the previously selected aptamers by qRT-PCR and flowcytometry (using fluorescently tagged aptamers) as previously described.37

These aptamers can also be evaluated for their ability to inhibit tumorcell line proliferation specifically. Aptamers with this property may bereceptor antagonists, which will be verified by examining their effecton cell signaling. Given the high homology between the human and mouseEphA2 extracellular domains (>90% identity; >90% structural homology),identifying aptamers that cross-react with human and mouse EphA2 can beas simple as testing the already selected aptamers for cross-reactivityagainst mouse. The existing set of 20 human EphA2 aptamers can thereforefirst be evaluated for the ability to bind mouse EphA2. Alternatively,the approach described above can be followed. For a few of the topaptamers, truncated sequences (lacking either or both of the libraryadapter sequences) can be synthesized to define the minimal sequencerequired for binding.

Aptamers of ˜20-35 nt in length can be identified for each ligand, whichcan be designed into AsiCs amenable for chemical synthesis.

In vitro assessment of TNBC-targeting AsiCs and their activity againstT-Ics. It can be defined which breast cancer subtypes are efficientlytransfected with TNBC-targeting AsiCs and evaluated whether tumorknockdown is specific relative to normal tissue cells, first in celllines and then in 10 tumor specimens to verify that the results for celllines translate to 10 tissues. We can also evaluate the potential ofTNBC-targeting AsiCs to transfect and target breast T-ICs.

AsiC design and initial testing The most attractive aptamers identifiedabove (prioritized based on considerations of affinity, selectivity ofbinding and expression in poor prognosis cancer vs normal cells,truncation to shorter length, the importance of the ligand inoncogenesis and stem cell behavior, receptor antagonism andcross-species reactivity) can be designed into AsiCs by linkage tosiRNAs targeting eGFP, AKT1 and PLK1 (vs control scrambled siRNAs) thathave been used for the initial EpCAM-AsiCs as described above herein.

Basal-like NBC cell lines stably expressing destabilized (dl)EGFP(protein T1/2 of −1 hr) were previously generated using lentiviruses.GFP expression can be readily quantified by flow and imaging, and itsknockdown has no biological consequences. The short T1/2 allows forrapid and sensitive detection of knockdown. AKT1, which is expressed inall cells, is a good endogenous gene to study, since its knockdown doesnot much affect cell viability.

PLK1 is used for its antitumor effect because its knockdown is cytotoxicto dividing cells. Described herein is robust and reproducible geneknockdown with EpCAM-AsiCs targeting each of these genes. AsiCs will bechemically synthesized with 2′-fluoropyrimidines for stability andinhibition of innate immune recognition and dT residues at their 3′-endsto protect against exonuclease digestion. The 2 strands will be annealedto generate the final RNA (FIG. 10A-10B). These AsiCs can be evaluatedand compared to the original EpCAM-AsiC (as positive control) and CD4-or PSMA-AsiCs (as negative control) in in vitro dose responseexperiments for AsiC uptake (using fluorophores such as AF-647 (whichdoesn't affect AsiC activity) conjugated to the 3′end of the shortstrand), gene knockdown and reduced tumor cell line growth and survival.Selective uptake, gene knockdown and antitumor effect in a few humanbasal-A TNBC cell lines (MB468, HCC1937, BPLER vs immortalizedepithelial cells) can be quantified by flow cytometry; flow cytometryand qRT-PCR; and Cell-TiterGlo and annexin-PI staining, respectively.These experiments can permit the selection of a handful of the bestperforming AsiCs that recognize EpCAM and EphA2.

Types of breast cancer responsive to TNBC-targeting AsiCs. It can bedetermined which types of breast cancer can be transfected with theselected AsiCs and how specific gene knockdown is in tumors relative tonormal epithelial cells. In vitro knockdown by the selected AsiCs in 20human breast cancer cell lines that represent the common breast cancersubtypes, but are weighted towards TNBC (14 TNBC lines, plus a samplingof luminal and Her2+ cell lines) can be evaluated. 93 Aptamer ligandexpression, uptake of fluorescent-labeled AsiC and gene silencing can becompared to BPE57 and fibroblast lines as negative controls. This largepanel of cell lines can permit evaluation of how cell surface EpCAM andEphA2 levels influence RNA uptake and gene silencing and whether thereis an expression threshold needed for efficient knockdown. A doseresponse experiment can permit verification that the high affinity ofthe aptamers is preserved in the AsiC. Specificity of uptake (versusnonspecific “sticking”) will be verified by using acid washing to removeloosely adhered aptamers and showing that binding is competed byunlabeled aptamers and eliminated when cells are trypsinized prior totreatment. AsiC-mediated transfection will be compared to lipidtransfection as positive control and to naked siRNA as negative control.Knockdown will be assessed by flow cytometry and qRT-PCR after 5 d, theoptimal time for AsiC-mediated knockdown. It is expected that uptake andgene silencing will correlate with aptamer ligand levels. To verify thatspecificity for tumor cells is maintained in mixtures of ligand+ andliganddim/− untransformed breast epithelial cells, we can comparefluorescent AsiC uptake, gene knockdown and survival when PLK1 is thegene target in mixtures of tumor cells expressing different aptamerligand levels with different numbers of GFP+ BPE cells.

Do epithelial primary breast cancer cells preferentially take upTNBC-targeting AsiCs and show knockdown relative to normal epithelialcells in tissue explants? To assess primary tumor uptake and knockdownand anticipate potential toxicity to normal tissue cells, we can nextassess in situ transfection and gene knockdown in explants of 10luminal, Her2+ and TNBC breast cancers and surrounding normal tissue. Wecan analyze samples from −25 tumors to provide a comprehensive look atcommon tumor subtypes. Tumor typing can be confirmed by histology andimmunohistochemistry (IHC) staining for ER, PR, Her2 and E-cadherin. Ifthe aptamer recognizes the mouse ligand, we can also assess potentialtoxicity to normal epithelia using mouse tumor/normal tissues. We cancompare normal tissues that have no large competing source of tumorcells to tissues that contain tumor cells. This might be important foranticipating toxicity in situations where AsiCs are given to patientswith low/undetectable tumor burden following therapy or surgery. Theseexperiments can also permit assessment of whether knockdown by 10 tumorsis comparable to that in cell lines, whether tissue architecture affectsuptake/knockdown in tumor cells and how well different tumor subtypesare transfected. It is contemplated herein that epithelial breastcancers will undergo efficient gene knockdown, but normal epithelialcells will not.

Biopsies, cut into −3×3×3 mm3 pieces, can be transfected in microtiterwells, which should mimic in vivo uptake after SQ or IV infusion.Lipofectamine encapsulated siRNAs and cholesterol-conjugated siRNAs areboth effective at gene knockdown of normal epithelial cells in polarizedcolumnar and squamous genital tract mucosa108,109, while naked siRNAsare not taken up. Similar results are expected with these controls innormal breast epithelial tissue. In parallel we can analyze knockdown ofcollagenase-digested 10 cells to compare knockdown with what is achievedin tissue and with cancer cell lines. We can first verify these controlsusing siRNAs to target epithelial genes, which we have previouslyknocked down (such as E-cadherin, cytokeratin (CK)-5 (a good marker ofbasal cells) and 14, and nectin-1) 93,108,109, whose expression can bereadily followed by IHC, fluorescence microscopy (FM) or flow cytometryof isolated cells. Staining of the target gene can be correlated withstaining for phenotypic markers and fluorescently labeled siRNAs todetermine which cell types are targeted. Pan-CK antibody can distinguishepithelial cells (normal and tumor) from stroma. Of particular interestis delivery and CK5 knockdown in rare basal tissue stem cells, sinceEpCAM-AsiCs can target these cells and potentially lead to depletion ofnormal tissue stem cells. Tissue toxicity and inflammation will beassessed by H&E staining of tissue sections and qRT-PCR assays for TypeI interferons and inflammatory cytokines (IL-1, IL-6, TNF-!). Additionalchemical modifications of the RNA sequence (besides2′-fluoropryrimidines) will be introduced to eliminate potentiallyharmful inflammation if it's detected.

Can TNBC-targeting AsiCs target breast tumor-initiating cells? We choseEpCAM and EphA2 as aptamer targets partly because of their potential totransfect T-ICs. Breast T-ICs are not uniquely defined by phenotypicmarkers (and they may in fact be heterogeneous93,110-113), makingexperiments challenging, since T-ICs are defined functionally by theirability to initiate tumors in small numbers that can be seriallytransplanted. Staining for CD44, CD24, EpCAM, CD133, CD49f or ALDH1 indifferent combinations enriches for T-ICs. 59,72,78,114-121 Differentprotocols define overlapping, but not identical, subsets of potentialT-ICs. Without wishing to be bound by theory, it is contemplated hereinthat EpCAM− and EphA2-AsiCs will be taken up by and cause gene silencingin T-ICs and can be used for targeted therapy to eliminate or crippleT-IC capability within tumors.

To analyze AsiC uptake and gene silencing in T-IC subpopulations,multicolor flow cytometry of EpCAM, EphA2, CD44 and CD24 in a panel ofbreast cancer lines (luminal, Her2+, basal-A and B TNBCs) can be used toidentify which breast cell lines have putative T-IC populations thatcontain cells that stain brightly for EpCAM and/or EphA2. We can alsoexamine EpCAM/EphA2 staining of mammospheres and Aldefluor+cells121,123-125 generated from these cell lines. We can select ˜4-5lines with the brightest/most uniform EpCAM/EphA2 expression withinT-ICs as the most attractive cell lines to study in this subaim and canproduce stable (dl)GFP-expressing variants. These cell lines, as well astheir mammospheres and Aldefluor+ subpopulation, can be incubated withAF647-labeled AsiCs (and as a negative control, nontargeting PSMA-AsiCs)bearing GFP siRNAs. AsiC uptake will be assessed by AF-647 fluorescencetogether with EpCAM or EphA2, CD44 and CD24 and Aldefluor staining.AsiCs can be taken up by EpCAM+ or EphA2+CD44+CD24-/dim Aldefluor+cells. To assess gene knockdown in T-IC phenotype cells, we can monitorGFP expression in the T-IC population and remaining cells by flowcytometry and qRT-PCR after treatment with eGFP or control siRNA-bearingAsiCs. We can also assess knockdown of endogenous PLK1 and AKT1.

These experiments can indicate whether T-Ics in different subtypes ofbreast cancer are targeted by EpCAM/EphA2-AsiCs. In subsequentexperiments we will focus on the cell lines in which we have >80%knockdown in T-IC-enriched populations. If knockdown is inefficient, wecan modify the transfection conditions (amount of AsiC, number of cells,volume, etc). Next, we can assess whether AsiCs inhibit mammosphere andcolony formation, reduce CD44 and ALDH1-expressing subpopulations, andthe size of the side population. In addition to knocking down PLK1, wecan design and evaluate AsiCs against a few additional genes that breastT-ICs depend on for self-renewal or maintaining multipotency. Basal-likeTNBC T-ICs are selectively sensitive to proteasome inhibition. 93

We can therefore evaluate knockdown of a proteasome component (PSMA2)and potentially other selective T-IC dependency genes (such as MSI1(Musashi), an RNA binding protein in breast T-ICs that regulates Wnt andNotch signaling126-130 or BMI1, a polycomb component required for stemcell self-renewal131-134). After verifying that these genes areexpressed and knocked down in mammosphere cells, we can treat bothadherent cells and mammospheres with AsiCs targeting PLK1, MSI1, BMI1 orPSMA2 or with AsiCs targeting eGFP as a negative control and measure thesize of T-IC subpopulations after 5-7 d by staining with CD44, CD24,EpCAM, CD133, CD49f and ALDH1. We can also measure the proportion ofcells that efflux small molecule dyes (the “side population”). Theseexperiments can be complemented by functional assays quantifying thefrequency of colony forming cells and mammospheres. Serial replating canassess whether the ability to continuously propagate T-ICs as spheres isinhibited. It is contemplated herein that knocking down PLK1, MSI1, BMI1or PSMA2 can reduce T-IC numbers, proliferation and function in theT-ICs from some cell lines, but different genes may be more active fordifferent breast cell lines. For example proteasome inhibitioneliminated T-ICs in basal-like TNBCs, but only in 1 of 3 mesenchymalTNBC cell lines and not in more differentiated non-TNBC tumors. 93

The knockdown approaches that suppress T-IC can be further investigatedby experiments using chemical inhibitors where available (such asbortezomib) or by examining whether knocking down other genes in thesame pathway (such as NOTCH1, 3-catenin or WNT1 for MSI1) also hasanti-T-IC activity. Next, we can determine whether short-term ex vivoexposure of basal-like TNBC lines to AsiCs inhibits TNBC tumorinitiation as the ultimate measure of inhibition of T-IC capacity, usingAsiCs that look promising in vitro. Cell lines, treated overnight withthe chosen AsiCs (and as negative controls AsiCs that use PSMA aptameror contain eGFP siRNA), will be assessed for viability. After verifyingthat short-term siRNA exposure does not affect viability, ex vivotreated cells will be injected in a range of cell numbers orthotopicallyinto NOD/scid/″c−/− (NSG) mice (these mice have the highest take fortumor implantation). Bortezomib treatment for 24 hr (at this time ˜40%of cells are still viable) can serve as a positive control.

In vivo evaluation of TNBC-targeting AsiCs

A few of the AsiCs that perform best can next be evaluated in vivo usingnude mice bearing mammary fatpad xenografts of an aptamer ligand+basal-A TNBC line, such as MB468 or HCC1187, on one side compared toligand-breast cancer cell line, such as basal-B MB231, on the other(˜5-8 mice/gp to obtain reproducible statistics based on our experiencewith these models). For in vivo imaging, we have already made stableluminescent/fluorescent cell lines by infection with luciferase- andmCherry-expressing lentivirus.

Systemic delivery and knockdown in tumor cells Because unmodified AsiCsare small (˜30 kDa), when injected IV or IP they are rapidly eliminatedby kidney filtration. 20 kDa polyethylene glycol (PEG) can be attachedto the 5′-end of the inactive (passenger) strand of the siRNA. 21 IVinjected PEGylated PSMA-AsiCs concentrated in subcutaneous tumors;PEGylation extended the circulating T1/2 of Ipinjected AsiCs from <35min to >>30 hr, increased the durability of gene silencing to ˜5 d andreduced the effective tumor-inhibitory dose 8-fold to 250 pmol×5injections. We have also found (not shown) that SQ injection of 5 mg/kgunmodified CD4-AsiCs caused systemic specific knockdown in CD4+ cells inthe spleen and proximal and distal lymph nodes of humanized mice.Therefore we can compare AsiC levels after IV and SQ administration ofthe original AsiC constructs and PEG-AsiCs by in vivo imaging usingAF-790-coupled AsiCs and the IVIS Spectrum and by Taqman assay of theactive strand in blood, urine, liver and tumor samples. Samples can beanalyzed over 5 d with frequent sample collection the first day. Tissuesections can be assessed for tissue damage and the blood can be analyzedfor hematological, liver and kidney toxicity by blood counts and serumchemistries. Toxicity associated with induction of innate immunity orinflammation can be assessed by ELISA assays of serum interferons andinflammatory cytokines. The circulating T1/2 and proportion of theinjected drug that localizes to the EpCAM+ tumor can be calculated.Based on our preliminary experiments with SQ and IV administration ofthe CD4-AsiCs and in vivo experience with the PSMA-AsiC9,21,25, it iscontemplated herein, without wishing to be bound by theory, thatunPEGylated AsiCs will be rapidly excreted after IV administration, butthat SQ EpCAM-AsiC and IV PEG-AsiCs will have more favorablelocalization to tumor xenografts.

Knockdown of mCherry and PLK1 following a single AsiC injection in arange of concentrations can be assessed by in vivo imaging and by flowcytometry, FM, and qRT-PCR of tumor specimens harvested 4, 7 and 12 dpost-treatment. These experiments can provide estimates of the effectivedose required for peak tumor gene knockdown of 50, 75 and 90% (ED50,ED75, ED90) and for the durability of knockdown in the tumor (quantifiedas T-KD50=time for tumor expression to return halfway to control fromthe peak knockdown). These parameters can be determined for eachconstruct. We can also determine the maximally tolerated dose (MTD) forthe PLK1 constructs. Inadequate PK/PD or signs of innate immunestimulation will lead us to adjust chemical modifications (adding 2′-OMeriboses to some residues) or add longer PEG polymers to improve theseparameters using straightforward.

Antitumor effect. It can be tested by in vivo imaging how effective thebest TNBC-targeting AsiCs are against basal-A tumors implanted in themammary fat pads or injected IV (as a metastasis model) in nude mice. Wecan begin by targeting PLK1 as proof of principle. 21,107 PLK1-AsiCs canbe injected SQ and/or IV in groups of 8 mice (group size chosen forstatistical significance based on previous experiments) bearing abasal-A TNBC fatpad tumor using dosing schedules chosen based on thePK/PD results. Mice can be treated as soon as tumors become palpable.Effects on a representative ligand+ and ligand-tumor will be compared.Control mice can be treated with PBS or naked siRNAs, AsiCs bearing ascrambled siRNA and PLK1 PSMA-AsiCs. Tumor size can be quantified byimaging and calipers. If the antitumor effect is suboptimal, the dosingregimen can be adjusted to the maximally tolerated regimen.

We can also compare the effect of PLK1 knockdown and standard-of-carechemotherapy, administered on their own and in combination to anticipatepotential clinical studies. If there is complete tumor regression, wecan evaluate decreased doses. Effective regimens can also be evaluatedin mice implanted with a few other basal-A TNBC lines to verify thegenerality of the antitumor response. We can also evaluate AsiCtreatment after tumor cells are injected IV to determine effectivenessagainst distal metastases. At the time of sacrifice, mice can besacrificed and mammary fatpads can be inspected for residual microscopicor macroscopic tumor by FM, H&E and IHC. Residual tumor cells can alsobe assessed for EpCAM/EphA2 expression to determine whether tumorresistance may have developed as a consequence of down-regulating theaptamers ligand. Treated mice can also be observed for clinical signs oftoxicity and at time of sacrifice can be carefully examined for gut andbone marrow toxicity, by blood counts and pathological examination ofgut, bone marrow and spleen. AsiCs designed with the cross-reactingaptamers can be used to evaluate normal epithelial toxicity. Using ourbest AsiC design, we can next begin to compare PLK1 knockdown withknockdown of TNBC dependency genes (such as PSMA2 or MCL1) identified inour siRNA screen93 tested alone or in combination with PLK1.

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Example 4

RNA interference (RNAi) offers the exciting opportunity to treat diseaseby knocking down disease-causing genes. Recent early phase clinicaltrials have shown promising and sustained gene knockdown and/or clinicalbenefit in a handful of diseases caused by aberrant gene expression inthe liver. The major obstacle to harnessing RNAi for cancer treatment isdelivery of small RNAs to disseminated cancer cells. Most epithelialcancer cells and the tumor-initiating cells (T-IC) within them highlyexpress EpCAM, the first described tumor antigen. All epithelial breastcancer cell lines we tested stain brightly for EpCAM, while immortalizednormal breast epithelial cells and fibroblasts do not. Targeted geneknockdown in epithelial cancer cells in vitro can be achieved usingchimeric RNAs composed of a structured RNA, called an aptamer, selectedfor high affinity binding to EpCAM, that is covalently linked to ansiRNA. These EpCAM aptamer-siRNA chimeras (AsiC) are taken up by EpCAM+cells and selectively cause gene knockdown in epithelial breast cancercells, but not normal epithelial cells. Moreover knockdown of PLK1 withEpCAM-AsiCs suppresses colony and mammosphere formation of epithelialbreast cancer lines, in vitro assays of tumor-initiating potential, andtumor initiation.

Subcutaneously injected PLK1 EpCAM-AsiCs are taken up specifically byEpCAM+ basal-A triple negative breast cancer (TNBC) orthotopicxenografts and cause rapid tumor regression. TNBC has the worstprognosis of any breast cancer and there is no targeted therapy for it.It is specifically contemplated herei that EpCAM-AsiCs can be used fortargeted gene knockdown to treat epithelial (basal-like) TNBC cancers,sparing normal cells, and eliminate the T-ICs within them. It can bedefined which breast cancer subtypes can be targeted by EpCAM-AsiCs anddetermine how EpCAM level affects uptake and gene silencing. Relativeuptake/knockdown in cancer cells expressing EpCAM and normal epitheliumcan be evaluated in human breast cancer tissue explants. It can also bedetermined whether EpCAM-AsiCs can target breast T-ICs to disrupt tumorinitiation.

The drug-like features of EpCAM-AsiCs can be optimized. EpCAM-AsiCs canbe optimized for cell uptake, endosomal release, systemic delivery andin vivo gene knockdown. Pharmacokinetics (PK) and pharmacodynamics (PD)of EpCAM-AsiC uptake and gene silencing and tumor suppression can beevaluated using live animal imaging in TNBC cell line xenograft models.As proof of principle, the antitumor effect of knockdown of PLK1, whichis needed for cell proliferation can be evaluated. In addition knockdownof novel gene targets identified in a genome-wide siRNA screen for TNBCgenetic dependencies will be evaluated in mouse xenograft models. Anoptimized EpCAM-AsiC and knowledge of its PK, PD and possible toxicity,can be used in experiments for further toxicity and other preclinicalstudies.

Described herein is the development of EpCAM aptamer-siRNA chimeras as amethod for targeted gene knockdown in basal-like triple negative breastcancer and other epithelial cancers and the tumor-initiating cellswithin them. There is currently no targeted therapy for triple negativebreast cancers, which frequently relapse, or for highly malignanttumor-initiating cell subpopulations within breast cancers, which may beresponsible for some cases of drug resistance and relapse. These RNAsprovide a versatile and flexible platform for RNA-based drugs to treatpoor prognosis breast cancers.

Example 5

It is demonstrated herein that (1) the EpCAM aptamer on its own does notaffect cell growth or viability of EpCAM+ breast tumor cell lines (notshown); (2) when normal breast biopsies are mixed with EpCAM+ TNBC humanbreast tumor tissues in vitro, fluorescent EpCAM-AsiCs only concentratein the tumor (FIG. 14); (3) treatment of EpCAM+ luminal and basal-A TNBCcells, but not mesenchymal TNBCs, with PLK1 EpCAM-AsiCs blocks in vitroassays of tumor-initiating cells (T-IC, colony and mammosphereformation) and in vivo tumor initiation (FIGS. 15A-15C and 16); (4)subcutaneously (sc) injected EpCAM− AsiCs concentrate in EpCAM+ tumorsin mice bearing EpCAM+ and EpCAM− TNBCs on either flank, distantlylocated from the injection site (FIG. 17A-18B); and (5) mostimportantly, sc injection of PLK1 EpCAM-AsiCs leads to completeregression of palpable basal-A TNBC xenografts (FIG. 18A-18B). Inaddition (6) a new siRNA screen identified novel shared geneticdependencies of basal-A TNBCs for EpCAM-AsiC knockdown (FIG. 19).

Without wishing to be bound by theory, T-ICs are heterogeneous andplastic in epithelial/mesenchymal gene expression. Although mesenchymaltraits may facilitate initial tissue invasion, formation of clinicallysignificant metastases (colonization) may require epithelial properties.EpCAM-mediated delivery of siRNA effectively blocks tumor initiation,but only for epithelial (basal-A TNBC, luminal) breast cancers.

The high affinity of the EpCAM aptamer and our uptake, gene knockdown,and proliferation experiments in uniform and mixed populations of cellsshow specific targeting to EpCAM+ cells. Normal epithelial cells andfibroblasts are not targeted. New data showing that EpCAM-AsiCs are nottaken up by normal human breast biopsies are compelling.

Triple negative breast cancer (TNBC), a diverse group of highlymalignant cancers that don't express the estrogen, progesterone and Her2receptors, has the worst breast cancer prognosis. There is no targetedtherapy for TNBCs, which often relapse after cytotoxic therapy.Described herein is a platform for gene knockdown therapeutics forbasal-like TNBC, using specifically targeted RNA interference (RNAi).RNAi can selectively knockdown disease-causing genes. Realizing thetherapeutic potential of gene knockdown for treating cancer, however,requires a robust method to deliver RNAs into disseminated cancer cells.There are 2 bottlenecks—getting RNAs across the cell membrane and fromendosomes to the target cell cytoplasm where the RNAi machinery sits. Anideal=therapy would selectively knockdown genes in cancer cells, whilesparing most normal cells to minimize toxicity.

Described herein is the knockdown of genes in basal-like TNBCs (themajority of TNBCs) with chimeric RNAs that use an aptamer (a structurednucleic acid selected for high affinity binding to a target moleculeagainst EpCAM (also known as CD326 or ESA)” +, the first described tumorantigen. EpCAM is highly expressed on epithelial breast cancers(including basal-like TNBC)—on average 400-fold more than on normalbreast tissue. It is also highly expressed on other epithelial cancersand is a marker of “cancer stem cells” (also called tumor-initiatingcells (T-IC)). Aptamer-siRNA chimeras (AsiC) covalently link a targetingaptamer to an siRNA (FIG. 10B). Dicer cleaves the siRNA from the aptamerinside cells.

Epithelial breast cancer cells, but not mesenchymal or normal epithelialcells, selectively take up EpCAM-AsiCs and undergo gene knockdown invitro. Moreover, knockdown strongly correlates with EpCAM levels.Knockdown of PLK1, a gene needed for mitosis, using EpCAM-AsiCseliminates colony and mammosphere formation (in vitro assays thatcorrelate with self renewal and tumor initiation) and tumor initiationin vivo, suggesting that EpCAM-AsiCs might be used to target T-ICs. Scinjection of PLK1 EpCAM-AsiCs caused complete regression of EpCAM+ TNBCxenografts, but had no effect on EpCAM− mesenchymal TNBCs.

It is described herein that EpCAM-AsiCs can be used for targeted geneknockdown to treat basallike TNBC cancers, sparing normal cells, andeliminate the T-ICs within them. Aside from their selective delivery totarget cells, AsiCs have important advantages for cancer treatmentcompared to RNA delivery by nanoparticles, liposomes or RNA-bindingproteins—(1) they bypass liver and lung trapping and concentrate intumors; (2) as a single RNA molecule they are simpler and cheaper tomanufacture than multicomponent drugs; (3) they have virtually notoxicity and do not stimulate innate immunity or inflammation or causesignificant off-target effects; (4) because they do not elicitantibodies, they can be used repeatedly; (5) they are stable in serumand other body fluids.

It can be defined which breast cancer subtypes can be targeted byEpCAM-AsiCs and determine how EpCAM level affects uptake and genesilencing. The relative uptake/knockdown in cancer tissues vs normalepithelium can be evaluated. It can also be determined whetherEpCAM-AsiCs can target breast T-ICs to inhibit tumor initiation. Animportant aim is to optimize EpCAM-AsiCs for uptake, endosomal release,systemic delivery and in vivo knockdown. Pharmacokinetics (PK) andpharmacodynamics (PD) of EpCAM-AsiC uptake, gene silencing and tumorsuppression will be evaluated by live animal imaging in TNBC orthotopicxenografts. As proof of principle, the antitumor effect of knockdown ofPLK1, which is needed for cell proliferation can be evaluated. Knockdownof other genes we identified in a genome-wide RNAi screen as geneticdependencies of basal-like TNBC can be evaluated. Described herein isthe development of optimized EpCAM-AsiC and knowledge of its PK, PD andpossible toxicity and identification of novel basallike TNBC dependencygenes to target

Described herein is: the verification of selective EpCAM-AsiC activityin epithelial breast cancers compared with normal epithelia and evaluatethe potential of EpCAM-AsiCs to transfect and eliminate breast T-ICs(i.e., cancer stem cells); optimization of EpCAM-AsiCs to transfect andknockdown genes in epithelial TNBC cells in vitro and for systemicdelivery and tumor concentration in vivo, and define PK and PD andmaximally tolerated dose; evaluation of the antitumor effect ofoptimized EpCAM-AsiCs targeting PLK1 and novel dependency genes ofbasal-like TNBC in human epithelial TNBC models of primary andmetastatic cancer in mice

Although most TNBC patients respond to chemotherapy, within 3 yr about athird develop metastases and eventually die. Thus we need newapproaches. TNBCs are heterogeneous, poorly differentiated tumors thatmay need to be treated by subtype or with individualized therapy.1,3,4,72 Most TNBCs are basal-like or belong to the basal-A subtype.Described herein is a flexible, targeted platform for treatingbasal-like TNBCs that is suitable for personalized therapy. Not onlywill the drug be targeted to the tumor, but the drug targets can also bechosen to attack the tumor's Achilles' heels by knocking down tumordependency genes. This present approach delivers small interfering RNAs(siRNA) into epithelial cancer cells by linking them to an RNA aptamerthat binds to EpCAM (FIG. 10B), a cell surface receptor over-expressedon epithelial cancers, including basal-like TNBCs. EpCAM is highlyexpressed on epithelial cancers and their T-Ics.

EpCAM targeting can cause selective gene knockdown in basal-like TNBCs,but not normal epithelia. Selective knockdown will both reduce the drugdose and reduce tissue toxicity.

As described herein, 9 of 9 basal-A TNBC and luminal breast cancer lineswere strongly EpCAM+, while a normal breast epithelial cell line,fibroblasts and mesenchymal TNBCs had close to background EpCAM (FIG.1B). Thus virtually all basal-like TNBCs (and probably luminal breastcancers) will be targeted by EpCAM-AsiCs. Moreover, since ˜100% ofepithelial cancers, including lung, colon, pancreas and prostate, stainbrightly for EpCAM, this platform could also be used for RNAi-basedtherapy of common cancers.

When RNAi was found in mammals, small RNAs were hailed as the next newdrug class. Soon investigators realized that getting RNAi to work as adrug was not simple., However, after addressing the main obstacle to RNAtherapy (cellular uptake), there is now optimism about RNAi-based drugs.Recent phase I/II studies have shown 80-95% gene knockdown inhypercholesterolemia, transthyretin-related amyloidosis, hepatitis C,hemophilia and liver metastasis, caused by aberrant liver geneexpression. However, applying RNAi for cancer therapy is still a dream.The major obstacle to harnessing RNAi for cancer is delivering smallRNAs into disseminated cells. Described herein are methods andcompositions that overcome this problem, e.g., by the use of AsiCs.

AsiCs are a flexible platform that can target different cell surfacereceptors and knockdown any gene or combination of genes. By changingthe aptamer, the AsiC platform can tackle the delivery roadblock thathas thwarted the application of RNAi-based therapy to most diseases.This approach is ideal for personalized cancer therapy, since the choiceof genes to target can be adjusted depending on a tumor's molecularcharacteristics. Moreover RNA cocktails can knockdown multiple genes atonce to anticipate and overcome drug resistance.

Described herein is the development of an optimized EpCAM-AsiC with welldefined PK/PD.

An important cancer research goal is to eliminate T-ICs (cancer stemcells). T-ICs are relatively resistant to chemotherapy and are thoughtresponsible for tumor relapse and metastasis The AsiCs described hereinare designed to target (epithelial) T-ICs with high efficiency. As suchthey can eliminate this aggressive subpopulation within tumors at riskfor progressive disease (see FIG. 16).

In addition to their potential therapeutic use, EpCAM-AsiCs can also bea powerful in vivo research tool for identifying the dependency genes oftumors and T-ICs to define novel drug targets.

Described herein is a novel targeted therapy for epithelial cancers, andthe T-ICs within them by targeting EpCAM, a tumor antigen widelyover-expressed in epithelial cancers and their T-ICs. Targeted therapyso far has relied on using tumor-specific antibodies or inhibitors tooncogenic kinases. No one before has shown that an unconjugated AsiC canhave potent antitumor effects or that AsiCs could be administered sc.There is currently no targeted therapy for TNBC or for T-ICs. Developingtargeted therapy for TNBC and developing ways of eliminating T-ICs areimportant unmet goals of cancer research.

The methods described herein are targeted in 2 ways—the aptamerspecifically delivers the therapeutic RNA to tumor cells, while thegenes chosen for knockdown can be selected based on the specificmolecular dependencies of the targeted tumor. By testing in vivoknockdown, it can be demonstrated that basal-like TNBCs and their T-ICsare selectively dependent on the proteasome, MCL1 and the U4/U6-U5tri-snRNP splicing complex. This work can identify a new set of drugtargets, suitable for both conventional and RNAi-based drugs.

The trafficking of siRNAs in transfected cells can be examined and eachstep of RNA processing in cells be systematically optimized to improvethe drug features of an siRNA.

CD4-AsiCs durably knockdown gene expression in CD4+T lymphocytes andmacrophages and inhibit HIV transmission to humanized mice. CD4-AsiCsspecifically suppressed gene expression in CD4+ T cells and macrophagesin polarized human cervicovaginal tissue explants and in the femalegenital tract of humanized mice. Because they are monomeric and don'tcross-link the receptor, CD4-AsiCs did not activate the targeted cells.They also did not stimulate innate immunity. Intravaginal application ofonly 80 pmol of CD4-AsiCs directed against HIV genes and/or CCR5 tohumanized mice completely blocked HIV sexual transmission. RNAi-mediatedgene knockdown in vivo lasted several weeks. Transmission was blocked byCCR5 CD4-AsiCs applied 2 d before challenge. Significant, butincomplete, protection also occurred when exposure was delayed for 4 or6 d. CD4-AsiCs targeting gag/vif provided protection when administeredpost-exposure. Thus CD4-AsiCs are promising for use in an HIVmicrobicide.

Protection against HIV transmission requires local knockdown in thegenital tract. However, systemic delivery is more challenging and isneeded for cancer. Because AsiCs are small enough to be filtered by thekidney, they are rapidly eliminated and do not efficiently cause genesilencing. In some embodiments, polyethylene glycol (PEG) can beattached to the 5′-end of the inactive (passenger) strand of the siRNA..iv injected PEG-AsiCs concentrated in sc tumors. PEGylation extendedthe circulating T1/2 of ip injected AsiC from <35 min to >>30 hr,increased the durability of gene silencing to ˜5 d and reduced theneeded dose 8-fold. sc injection of unmodified CD4-AsiCs caused ˜80%gene knockdown specifically in CD4+ cells in the spleen, proximal anddistal lymph nodes of humanized mice (not shown). Sc injection ofEpCAM-AsiCs similarly led to specific concentration/knockdown in EpCAM+tumors (see below).

EpCAM-AsiCs selectively knockdown gene expression in EpCAM+ cancer cellsThe EpCAM-AsiCs have a ˜42-44 nt long strand (19 nt aptamer+linker+20-22nt siRNA strand) annealed to a 20-22 nt complementary siRNA strand (FIG.10B). Commercially synthesized with 2′-fluoropyrimidines, they are RNaseresistant (T1/2>3 d in serum, data not shown) and do not trigger innateimmunity. 37,91-93

Surface EpCAM was high in all luminal and basal-like cell lines tested,but close to background in normal epithelia immortalized with hTERT(BPE) 94, fibroblasts and mesenchymal TNBCs (FIG. 1B). Several of ahandful of designs tested (with the sense and antisense strandsexchanged and several linkers) knocked down gene expression specificallyin EpCAM+ cell lines, but the most effective design is shown in FIG.10B. Gene knockdown of eGFP and AKT1 by EpCAM-AsiCs was uniform andselective for EpCAM+ cells and as effective as siRNA lipid transfection,which was not selective (FIG. 13A-13C). In 8 breast cancer cell lines,AKT1 knockdown and inhibition of cell proliferation by PLK1 EpCAM-AsiCsstrongly correlated with EpCAM levels (FIG. 11B-11C). The EpCAM aptameron its own had no effect on cell proliferation (not shown). When EpCAM−BPE cells were mixed with epithelial TNBC cell lines, EpCAM-AsiCsknocked down AKT1 and caused PLK1-sensitive cell death only in tumorcells, sparing the normal epithelial cells (not shown). The proportionof surviving tumor cells decreased 7-fold after 3 d. When we addedfluorescent AsiCs, cholesterol-conjugated siRNAs (chol-siRNA, taken upby normal epithelia) or naked siRNAs to normal breast and tumor biopsysamples, EpCAM-AsiCs concentrated only in the tumors (FIG. 14). ThusEpCAM-AsiCs are specific for epithelial tumor cells.

EpCAM-AsiCs inhibit T-ICs of EpCAM+ tumors. EpCAM was chosen fortargeting partly because EpCAM marks T-ICs and metastasis-initiatingcells (M-IC). To investigate whether EpCAM-AsiCs inhibit T-ICs, wecompared colony and mammosphere formation (T-IC functional surrogates)after mock treatment, treatment with paclitaxel or with EpCAM-AsiCsagainst eGFP or PLK1. PLK1 EpCAM-AsiCs more strongly inhibited colonyand mammosphere formation of multiple EpCAM+ basal-like TNBCs and aluminal cell line than paclitaxel, but was inactive against EpCAM−basal-B TNBCs (FIG. 15A-15C). To evaluate EpCAM-AsiC's effect on tumorinitiation, viable luc+ EpCAM+ MB468 and EpCAM− MB231 cells, treatedovernight with medium or PLK1 or GFP EpCAM-AsiCs, were implanted sc innude mice. PLK1 EpCAM-AsiCs blocked tumor formation, but only in EpCAM+tumors (FIG. 16 and data not shown). Thus EpCAM-AsiCs inhibit tumorinitiation in EpCAM+ breast cancers.

EpCAM-AsiCs are selectively taken up by EpCAM+ TNBCs and cause tumorregression To investigate the potential clinical usefulness ofEpCAM-AsiCs, we first examined delivery of Alexa750-labeled EpCAM-AsiCsinjected sc in the scruff of the neck of mice bearing EpCAM+ and EpCAM−TNBCs in each flank (FIG. 17A-17B). EpCAM-AsiCs concentrated only in theEpCAM+ tumor. Mice bearing bilateral tumors were mock treated orinjected biweekly with PLK1 or GFP EpCAM-AsiCs and tumor growth wasfollowed by luminescence. The EpCAM+ tumors rapidly completely regressedonly in mice that received the PLK1-targeting AsiCs (FIG. 18A-18B). Thisexperiment was repeated with additional control groups, the EpCAMaptamer on its own or PLK1 siRNA, neither of which had any anti-tumoractivity (data not shown). Thus sc injected EpCAM-AsiCs show specificantitumor activity against basal-A TNBCs.

Live cell imaging of siRNA uptake, endosomal release and gene silencingAn optimized spinning disk confocal microscope capable of singlemolecule detection was used to detect the weak cytosolic signal ofreleased fluorescent RNAs, which was not before possible. HeLa cellsincubated with Alexa647-siRNA lipoplexes were imaged every 3 s.RNA-containing late endosomes released a small fraction of their cargoRNA, which diffused rapidly to fill the cytosol (data not shown).Release occurred during a narrow time frame, ˜15-20 min afterendocytosis. ˜104 siRNAs were released in a typical event. In HeLacells, stably expressing eGFP-dl, GFP siRNAs caused GFP expression todecrease rapidly after endosomal release with a T1/2 of ˜2.5 h. Only˜1000 cytosolic siRNAs were needed for efficient gene silencing. Releasetriggered autophagy, which sequestered the RNA-containing endosomewithin a double autophagic membrane. No release occurred after that.

We applied this method to study uptake/release of Cy3-labeledEpCAM-AsiCs, comparing EpCAM+ MB468 TNBCs with EpCAM− BPE cells. Uptakeand release were negligible in BPE, but clear cut in MB468. This imagingmethod and our understanding of siRNA trafficking can be used tooptimize EpCAM-AsiC design to improve endosomal release and knockdown.

Identification of basal-like TNBC dependency genes (BDGs). To identifygenetic dependencies of basal-like TNBCs that EpCAM-AsiCs could target,a genomewide siRNA lethality screen was performed comparing basal-likeBPLER and myoepithelial HMLER cells, human primary breast epithelialcells transformed with the same oncogenes in different media. Althoughessentially isogenic, BPLER are highly malignant and enriched for T-ICs,forming tumors in nude mice with only 50 cells, while HMLER require >105cells to initiate tumors. The screen identified 154 genes on whichBPLER, but not HMLER, depended. Proteasome genes were highly enriched(P<10-14). Expression of BPLER dependency genes correlated with poorprognosis in breast, but not lung or colon, cancer. Proteasome inhibitorsensitivity was a shared feature of basal-A TNBCs and correlated withMCL1 dependency. Normal breast epithelial cells, luminal breast cancerlines and mesenchymal TNBC lines did not depend on the proteasome orMCL1. Proteasome inhibition not only killed basal-A TNBCs, it alsoblocked T-IC function by colony and mammosphere assays, again mostlyselectively in basal-like TNBCs. Brief exposure to bortezomib alsoinhibited tumor initiation of a mouse basallike TNBC line.

We next tested whether proteasome inhibition inhibited the growth ofbasal-like TNBC tumors in mice. Bortezomib does not penetrate well intosolid tumors, which has limited its clinical use. The maximum toleratediv dose (MTD) was needed to inhibit proteasome activity in sc tumors.Treatment with the MTD strongly inhibited tumor growth of 3 human and 1mouse basal-A TNBC cell lines and 10 TNBCs that arose spontaneously inTp53+/− mice, but was not active against basal-B or luminal cell lines.Similar results were obtained with carfilzomib. Bortezomib also blockedlung colonization of iv-injected mouse TNBC cells. Thus the proteasomeis selectively required for epithelial TNBC growth, tumor initiation andmetastasis. Although tumor penetration and PD may improve with newerproteasome inhibitors, proteasome gene knockdown might provide moreeffective proteasome inhibition.

Because TNBCs are heterogeneous1,3,4,72, we rescreened the 154 BPLERdependency genes in 4 basal-A TNBC and 3 luminal human cancer lines. Ourgoal was to identify additional shared dependencies of basal-like TNBCcell lines as potential EpCAM-AsiC targets. Only 21 of the 154 BPLERdependency genes reduced viability by at least 2-fold in 3 of 4 basal-Acell lines tested. These putative BDGs clustered in 4 functionalgroups—4 proteasome genes and MCL1 (previously validated), 10 genesimplicated in RNA splicing, 2 genes implicated in mitosis and 2 genesrequired for nuclear export. 20 of the 21 BDGs genes were retested usinga new set of siRNAs and 14 genes reconfirmed (the other “hits” may havebeen secondary to off-target effects or their knockdown could have beeninsufficient to cause lethality). Of note, 9 of 10 splicing genesreconfirmed. They included 4 members of the U4/U6-U5 tri-snRNP complex,PRPF8, PFPF38A, RBM22, USP39. Other interesting shared hits were the RANnuclear export G protein and the nucleoporin NUP205, and NDC80, akinetochore component that anchors the kinetochore to the mitoticspindle. (USP39 is also required for the mitotic spindle checkpoint).

TNBCs are known to be particularly susceptible to antimitotic agents.USP39 is overexpressed in breast cancer cells vs normal breast tissueand USP39 knockdown inhibited proliferation and colony formation ofluminal MCF7 cells. Moreover in zebrafish, USP39 mutation leads tosplicing defects of tumor suppressor genes like rb1 and p21. To explorethe therapeutic effect of inhibiting splicing in basal-like TNBCs, wesilenced the 4 spliceosome tri-snRNP complex BDGs (PRPF8, PRPF38A,RBM22, USP39) in 6 basallike cell lines and in luminal MCF7 cells (FIG.19). Knock down of PRPF8, PRPF38A or RBM22 activated caspase-3 and waslethal for 6 of 6 basal-like cell lines, but not for MCF7; USP39knockdown killed 3 of 6 basal-like cell lines. Spliceosome proteins werefrequently up regulated in breast cancer cell lines of all subtypes. Theviability of all 6 basal-like cells lines, but not MCF7 cells, wasreduced at least 2-fold by knockdown of the mitotic kinetochore geneNDC80 or of nuclear export genes RAN or NUP205. Moreover, knockdown ofeach of the tri-snRNP complex genes, RAN, NUP205 or NDC80 blocked colonyformation (a surrogate of T-IC potential) in 3 of 3 basal-like TNBC celllines

EpCAM-AsiCs can cause targeted gene knockdown in EpCAM+ tumors and theT-ICs within them. Although there may be some uptake in normalepithelial cells that weakly express EpCAM, gene knockdown will beconcentrated in EpCAMbright tumor cells, especially in T-ICs.EpCAM-AsiCs can be optimized, as described herein, for favorable PK/PDto suppress tumor growth and metastasis of basal-like TNBCs withacceptable toxicity in mouse models.

EpCAM-AsiCs targeting eGFP, AKT1 and PLK1 are used herein as models forassessing gene knockdown and optimizing AsiC design. Cell lines stablyexpressing destabilized (dl)EGFP, with a protein T1/2 of −1 hr, can begenerated using lentiviruses. GFP expression can be readily quantifiedby flow and imaging, and its knockdown has no biological consequences.The short T1/2 allows for rapid and sensitive detection of knockdown.AKT1, which is expressed in all the cells we test, is a good endogenousgene to study, since its knockdown in TNBCs doesn't affect cellviability much. PLK1 is used as proof-of-concept for its antitumoreffect because its knockdown is cytotoxic to all dividing cells. Wepreviously showed that PLK1 knockdown using a different deliverystrategy dramatically suppressed Her2+ breast cancer in mice. In arecent screen, PLK1 was unique amongst kinase genes because itsknockdown eliminated breast T-ICs. We have achieved robust andreproducible gene knockdown with EpCAM-AsiCs targeting each of thesegenes.

EpCAM-AsiCs can be be purchased, e.g., as non-GMP RNAs from TriLink orNITTO Avecia. Each strand of the EpCAM-AsiC was synthesized with2′-fluoropyrimidines and dT residues at their 3′-ends to protect againstexonuclease digestion and then annealed to generate the final RNA (FIG.10B). As we optimize the AsiC, other chemical modifications can besubstituted and tested to determine if they confer improved activity.The aptamer alone and AsiCs bearing a nontargeting siRNA can serve ascontrols. Some of the eGFP EpCAM-AsiCs can also be annealed to anantisense strand modified at the 3′-end with a fluorophore (whichdoesn't affect AsiC activity (not shown)) to quantify AsiC uptake andtrafficking within cells and in vivo.

Specific EpCAM-AsiC knockdown in epithelial breast cancers and breastcancer T-ICs vs normal epithelial cells. It can be determined whichbreast cancer subtypes are transfected with EpCAM-AsiCs and evaluatewhether tumor knockdown is specific to cancer cells, first in cell linesand then in 10 tumor tissues to verify that the results for cell linestranslate to tissues in situ. Because EpCAM-AsiCs might also transfectnormal tissue stem cells, knockdown and toxicity to these rare basalcells will be assessed in the tissue experiments. We can also evaluatethe potential of EpCAM-AsiCs to transfect and target breast T-ICs.

Types of breast cancer responsive to EpCAM-AsiCs We first need to knowwhich types of breast cancer can be transfected with EpCAM-AsiCs and howspecific gene knockdown is in tumors relative to normal epithelialcells. We extend our prelim. studies (FIGS. 13A-13C and 11B-11C) byevaluating in vitro knockdown in a panel of 20 human breast cancer celllines that represent the common breast cancer subtypes, but are weightedtowards TNBC (14 TNBC lines, plus a sampling of luminal and Her2+ celllines). 95 EpCAM expression, uptake of Cy3-labeled AsiC and genesilencing in tumor lines can be compared to that in BPE94 andfibroblasts. This large tumor panel will enable us to evaluate how cellsurface EpCAM levels influence gene silencing and whether there is anEpCAM expression threshold for efficient knockdown. We can also verifyin a dose response experiment using a few EpCAM+ cell lines that thereported high binding affinity of the EpCAM aptamer is preserved in theAsiC. Specificity of uptake (versus nonspecific “sticking”) can beverified by using acid washing to remove loosely adhered aptamers andshowing that binding is competed by unlabeled aptamers and eliminatedwhen cells are trypsinized before treatment. EpCAM-AsiC-mediatedtransfection can be compared to lipid transfection and naked siRNAs ascontrols. Knockdown will be assessed by flow cytometry and qRT-PCR after5 d, the optimal time for AsiC-mediated knockdown. We expect that uptakeand gene silencing will correlate with EpCAM levels. To verify thatspecificity for EpCAM+ cells is maintained in mixtures of EpCAM+ andEpCAMdim untransformed breast epithelial cells, we can comparefluorescent EpCAM-AsiC uptake, gene knockdown and survival when PLK1 isthe gene target in mixtures of tumor cells expressing different levelsof EpCAM (MFI ranging between 100-1000) with different numbers of GFP+BPE cells.

Do epithelial breast cancer cells preferentially take up EpCAM-AsiCs andshow knockdown relative to normal epithelial cells in tissue explants?To assess primary tumor knockdown and anticipate potential toxicity tonormal tissue cells, we can assess in situ transfection and geneknockdown in explants of 10 luminal, Her2+ and TNBC breast cancers andsurrounding normal tissue from mastectomy specimens. Samples from −25tumors can be analyzed to provide a comprehensive look at tumorsubtypes. Tumor typing can be confirmed by histology and IHC stainingfor ER, PR, Her2, E-cadherin. We can compare normal tissues that have nolarge competing source of EpCAM+ cells to tissues that contain tumorcells. This might be important for anticipating toxicity in situationswhere AsiCs are given to patients with low/undetectable tumor burdenfollowing therapy or surgery. These experiments can permit theassessment of whether knockdown by 10 tumors is comparable to that incell lines, whether tissue architecture affects uptake/knockdown intumor cells and how well different tumor subtypes are transfected.

Based on the data presented herein, e.g., FIG. 14, it is contemplatedherein that epithelial breast cancers, but not normal epithelial cells,can undergo efficient gene knockdown. Tissues cut into −3×3×3 mm3samples can be transfected in Optimem solution in microtiter wells.Lipoplexed siRNA and chol-siRNAs both knockdown genes in normal columnarand squamous genital tract epithelia, while naked siRNAs are not takenup. We can first verify these controls using siRNAs to target epithelialgenes, which we have previously knocked down (such as E-cadherin,claudin3, cytokeratin (CK)-5 (a good marker of basal cells), andnectin-1), whose expression can be readily followed by IHC, fluorescencemicroscopy (FM) or flow cytometry of separated cells. Staining of thetarget gene product can be correlated with staining for phenotypicmarkers and fluorescent siRNAs to determine which cell types within thetissue are targeted. Pan-CK antibody can be used to distinguishepithelial cells (normal and tumor) from stroma. We can also compareknockdown of collagenase-digested 10 cells to tissue knockdown. Withoutwishing to be bound by theory, delivery and CK5 knockdown in rare basaltissue stem cells can be assessed, since EpCAM-AsiCs may target thesecells and potentially lead to toxicity. Because toxicity to the GI tractis often dose limiting for cancer drugs, we can repeat these studiesusing colon tumor specimens to determine whether colon cancer cells,normal gut epithelia and crypt stem cells are transfected. Theseexperiments can provide useful data regarding clinical toxicity and thechoice of genes to knockdown, i.e. we might knockdown cancer dependencygenes that are not essential for normal stem cells, if tissue stem cellsare efficiently transfected. (Hematopoietic cells don't express EpCAM,so hematological toxicity is not expected.)

Can EpCAM-AsiCs be used to target breast tumor-initiating cells? Onereason we chose EpCAM as aptamer target is its potential to transfectT-ICs (“cancer stem cells”). T-ICs are drug resistant and thoughtresponsible for tumor initiation, relapse and metastasis. Breast T-ICsare not uniquely defined by phenotype, making experiments challenging,since T-ICs are defined functionally by their ability to initiate tumorsthat can be serially transplanted. Staining for CD44, CD24, EpCAM,CD133, CD49f or ALDH1 in different combinations enriches for T-ICs.49,61,67,107-111

Different protocols define overlapping, but not identical, subsets ofpotential T-ICs. T-ICs are heterogeneous and show plasticity in theirepithelial vs mesenchymal features (and in fact may have some featuresof both states). 28,95,112-118 Some breast T-ICs are mesenchymal anddon't express EpCAM. However, there is increasing evidence that theability of basal-like TNBCs to colonize distant tissues and formmacroscopic metastases—arguably the most clinically important functionof T-ICs—depends on epithelial properties. Moreover our new data (FIGS.15A-15C and 16) on the effect of EpCAM-AsiCs on T-IC function and tumorinitiation indicate that EpCAM-AsiCs have anti-T-IC activity for basal-ATNBCs. We hypothesize that EpCAM-AsiCs are taken up by basal-like TNBCT-ICs and can be used for targeted therapy to cripple T-IC capabilitywithin them.

To analyze EpCAM-AsiC uptake and gene silencing in T-ICs, we can firststain a panel of breast cancer lines with EpCAM, CD44 and CD24 toidentify breast cell lines whose putative T-IC populations contain cellsthat stain brightly for EpCAM. We can also examine EpCAM staining ofmammospheres and Aldefluor+ cells 111,123,124 generated from these celllines. We can select ˜4-5 lines with the most uniform EpCAM expressionwithin T-ICs as the most attractive cell lines to study in this subaim(and as controls, 1-2 basal-B cell lines whose T-ICs might lack EpCAMstaining) and can produce stable eGFP-expressing variants. These celllines, and their mammospheres and Aldefluor+ subpopulation, can beincubated with fluorescent eGFP EpCAM-AsiCs (and as control,nontargeting PSMA-AsiCs). AsiC uptake can be assessed together withEpCAM, CD44 and CD24 and Aldefluor staining. AsiCs should be taken up byEpCAM+CD44+CD24−/dim Aldefluor+ cells. To assess gene knockdown in T-ICphenotype cells, we can monitor GFP in the T-IC population and remainingcells after treatment with eGFP or control siRNA-bearing AsiCs by flowcytometry and qRT-PCR (of Aldefluor+ or mammosphere populations). We canalso assess knockdown of endogenous PLK1 and AKT1. These experiments cantell us whether T-ICs in different subtypes of breast cancer aretargeted by EpCAM-AsiCs. Next we assess whether AsiCs inhibitmammosphere and colony formation, reduce phenotypic T-IC subpopulations,or the side population.

We can also design and evaluate AsiCs against additional genes neededfor self-renewal or multipotency. Because basal-like TNBC T-ICs aresensitive to proteasome inhibition, we can evaluate knockdown of aproteasome component (PSMA2). Other potential T-IC dependency genes wewill evaluate are MSI1, a gene highly expressed in breast T-ICs thatregulates Wnt and Notch signaling 125-129, BMI1, a polycomb componentneeded for self-renewal 130-133, and possibly a few novel BDGsidentified in our recent siRNA screen (FIG. 19). MSI1 knockdowndecreases stem cell markers and mammosphere formation in MCF7 and T47Dcells. 129

After verifying that these genes are expressed and knocked down inmammosphere cells, we can treat both adherent cells and mammosphereswith AsiCs targeting these genes or eGFP as a negative control andmeasure the size of T-IC subpopulations after 5-7 d by staining forCD44, CD24, EpCAM, CD133, CD49f and ALDH1. We can also measure theproportion of cells that efflux small molecule dyes (the “sidepopulation”). These experiments can be complemented by functional assaysquantifying colony forming cells and mammospheres. Serial replating caninvestigate whether propagation of T-ICs as spheres is inhibited.

Knocking down PLK1, MSI1, BMI1 or PSMA2 can reduce T-IC numbers,proliferation and function in some breast cancer subtypes, but differentgenes may be more active for different breast cell lines (i.e.proteasome inhibition eliminated T-ICs in basal-like TNBCs, but notnon-TNBC tumors and in only 1 of 3 basal-B TNBCs95). The knockdownapproaches that suppress T-IC can be further investigated by experimentsusing available chemical inhibitors and/or by knocking down other genesin the same pathway (such as NOTCH1, β-catenin or WNT1 for MSI1). Theeffect on T-ICs of EpCAM-AsiCs can be compared with the EpCAM aptamer onits own and the EpCAM antibody, adecatumumab (Amgen).

Next we determine whether short-term ex vivo exposure of basal-like TNBClines to EpCAM-AsiCs inhibits tumor initiation as the ultimate measureof T-IC inhibition. The most promising AsiCs can be tested in vivo. Celllines, treated overnight with AsiCs (and as negative controls AsiCs thatuse PSMA aptamer or contain eGFP siRNA), can be assessed for viability.After verifying that short-term siRNA exposure does not affectviability, ex vivo treated cells will be injected in a range of cellnumbers orthotopically into NOD/scid/!c−/− (NSG) mice (these mice havethe highest take for tumor implantation). Pretreatment with bortezomib,which reduced tumor initiation in basal-like TNBC”), or adecatumumabwill be controls.

Optimize EpCAM-AsiCs To improve EpCAM-AsiC drug features, we canoptimize each step of in vitro gene knockdown and in vivo delivery. Wecan also modify the chemistry of EpCAM-AsiCs (if needed) to minimizeoff-target effects.

In prelim. studies and published work, the AsiC concentration needed foroptimal knockdown in vitro is ˜1-4 μM, many fold higher than the −100 nM(or lower) concentrations used for lipid transfection. For knockdown,EpCAM-AsiCs follow the following steps: (1) cell receptor binding, (2)endocytosis, (3) endosomal release, (4) Dicer processing, (5)incorporation into the RNA-induced silencing complex (RISC), and (6)target mRNA cleavage. We can systematically optimize each step, focusingon steps (2) and (3), where we expect we can obtain the largest gains inefficacy. The AsiC design variables are the EpCAM aptamer, whoseaffinity affects steps 1 and 2; the linker sequence between the aptamerand the siRNA, which controls step 4; the siRNA sequence, which controlsstep 6. In addition each residue used for chemical synthesis fromphosphoramidite building blocks can be chemically modified to reducenuclease digestion, off-target suppression of partially complementarysequences, binding and stimulation of innate immune RNA sensors andimprove cell uptake and in vivo PK. The most common chemicalmodifications are substituting S for O in the phosphate backbone (toproduce RNase-resistant phosphorothioate (PS) linkages and substituting2′-F, 2′-O-methyl (2′OMe), or 2′-O-methyoxyethyl (2′MOE) for the 2′-OHin the ribose. PS, 2′-F and 2′-OMe modifications are well tolerated inclinical trials and therefore we concentrate on them. 2′-OMe occursnaturally in rRNA and tRNA and is therefore safe, and 2′-F is also welltolerated; heavily Psmodified nucleotides are sticky (and cause bindingto serum proteins, which can improve circulating T1/2) and can causeunwanted side effects; lightly modified PS-RNAs are not toxic. Chemicalmodifications can both inhibit and enhance gene silencing in steps 5 and6 This can be an iterative process; as modifications are made at onestep, the most attractive modified candidates can be optimized for othersteps, drawing on lessons learned from previous candidates. We canverify that the modified AsiCs chosen for further development do notstimulate innate immunity or result in cellular toxicity. If they do, wecan further modify our designs to avoid these problems.

Optimize In Vitro Knockdown

(1) EpCAM binding The EpCAM aptamer has 12 nM affinity, It can beverified that that this affinity is preserved in the EpCAM-AsiC. If theAsiC has lower affinity than the aptamer, we can use bio-layerinterferometry (OctetRED System, ICCB-Longwood Core) with recombinantEpCAM to compare the affinity of the aptamer and AsiC. If the AsiC haslower binding affinity, it may not fold properly. To enhance foldinginto the desired conformation we can try changing the type and length ofthe linker between the aptamer and the AsiC sense strand (i.e. we canincorporate more 3C linkers or triethylene or hexaethylene glycolspacers).

(2) Endocytosis The monomeric AsiC is slowly taken up by constitutivereceptor recycling. This step can be optimized by receptor crosslinkingto trigger active endocytosis, which requires aptamer multimerization.Multimerization of aptamers (with or without linked siRNAs) can increasebinding avidity (by increasing valency) or convert an aptamer that doesnot cause signaling into an agonistic reagent. Aptamers can bemultimerized by using streptavidin (SA) to bind biotinylated (Bi)aptamers and siRNAs; extending the aptamer with an adapter that binds toan organizing oligonucleotide that contains multiple complementarysequences connected by a flexible linker; or extending the aptamer withcomplementary adapter sequences to produce a dimer. We focus on all-RNAdesigns, which don't induce antibodies. Some of the designs we can testare shown in FIG. 20 (we can also test constructs with sense andantisense strands exchanged).

Time course and dose response experiments will compare fluorescentlytagged multimeric constructs with the monomeric AsiC to assess theextent and rapidity of uptake and GFP knockdown by flow cytometry andlive cell imaging (data not shown). If endocytosis is enhanced bymultimerization, but knockdown does not improve, we can use Northernblotting to follow Dicer cleavage and determine whether the expectedantisense strand is produced (see below). If not, we can alter thedesign of the linkers, for example by lengthening the duplex region from21 to 27 nt, so the multimerized AsiC is a good Dicer substrate(&) andverify that the 5′ end of the Dicer product originates at the intendedbase. Multimerization should reduce the AsiC concentration needed forknockdown many fold. However, multimerization could cause unwanted EpCAMsignaling and promote tumor cell proliferation. We can verify that thisis not the case using multimerized constructs targeting eGFP. Anattractive feature of multimerization is that it could link multipledifferent siRNAs into a single RNA molecule for combinatorial geneknockdown to produce a cancer “cocktail”.

If none of these multimers work, we can test monomeric AsiCs containingcomplementary sequences that enable RNAs to selfassemble into smallnanoparticles or the SA-Bi strategy, using less immunostimulatory SAmutants.

(3) Endosomal release Although fewer than 1000 cytosolic siRNA moleculesare estimated to be needed for knockdown (not shown), only a few percentof siRNAs in endocytosed liposomes are released into thecytosol.EpCAM-AsiC endosomal release can be assessed by live cell imaging tomeasure the efficiency of cytosolic release of endocytosed AsiCs. Ifthis indicates less than desired endosomal release, then improvingrelease should reduce the drug dose substantially. Preincubation andendocytosis of an amphipathic cationic peptide (mellitin) or polymer(butyl vinyl ether) that is reversibly masked, can enhance siRNA escapeto the cytosol. Masking means that at neutral pH the peptide or polymeris uncharged and does not interact with the plasma membrane and damageit, but at the negative endosomal pH, a cationic molecule is generatedthat damages the endosomal membrane and releases coendocytosedoligonucleotides. Iv injection of these masked polymers within 2 hr ofsiRNA delivery potentiated hepatocyte knockdown by chol-siRNAs as muchas 500 fold in mice and nonhuman primates.

We can first determine by live cell videomicroscopy whether priortransfection of masked cationic polymers facilitates EpCAM-AsiC (andlipoplexed siRNA) cytosolic delivery and eGFP knockdown in vitro. We canalso investigate whether incubating EpCAM-AsiCs with basicpeptides/polymers can also determine whether inhibition of endosomalacidification using bafilomycin A or concanamycin alters EpCAM-AsiCcytoplasmic release and knockdown, as the proton sponge theory predicts.If these experiments confirm the proton sponge theory, we caninvestigate strategies for altering EpCAM-AsiCs. These include covalentconjugation (via disulfide bonds spontaneously reversed in the cytosol'sreducing environment) of the sense or antisense strand to cellpenetrating peptides, including polyarginines of different sizes,protamine152, mellitin, transportan or penetratin and conjugation of theAsiC sense strand to butyl and amino vinyl ester or linkage of the sensestrand to phosphospermines of different lengths. We can verify thatthese modifications do not alter solubility, result in cytotoxicity orinnate immune stimulation or interfere with specific EpCAM targeting.

Dicer processing, RISC incorporation, target mRNA cleavage We next takethe top 2-3 EpCAM-AsiCs, with the initial design as control, and examinewhether siRNA function can be optimized. Northern blots, probed for thesense, antisense and aptamer parts of the EpCAM-AsiC, can analyzeEpCAM-AsiC products within cells. Their migration can be compared tothat of synthesized sense and antisense strands, aptamer and full lengthEpCAM-AsiC. If Dicer cleaves the AsiC as expected, we can recover RNAsthat migrate like the sense and antisense strands (as well asunprocessed EpCAM-AsiCs from endosomes and a band the size of theaptamer joined to its linker). (Dicer dependence can be verified usingHCT116 cells expressing hypomorphic Dicer). If the intracellular RNAsare not the expected size, we can clone them to determine where Dicercuts. If the bands are not cut or are not where we want, we can redesignthe linker and double stranded region to produce the desired cleavage.We can also investigate replacing the UUU linker with alternativelinkers or combinations of linkers, by substituting or adding one ormore 3C linkers or triethylene or hexaethylene glycol spacers, toenhance intracellular processing to the siRNA. We can also investigatewhether a Dicer-independent design in which the aptamer is covalentlyjoined to the sense or antisense strand of the siRNA by a disulfidebond, spontaneously reduced in the cytosol, leads to more efficientknockdown.

Once we have shown that the appropriate antisense strand is produced, wecan next compare antisense strand incorporation into the RISC. Northernblotting and Taqman PCR will quantify how much of the input activestrand in whole cell lysates is pulled down with pan-Ago antibody (2A8).Ago binding, the T1/2 of the siRNA in the RISC, and target geneknockdown are influenced by chemical modifications of the sense andantisense strands. Specific 2′-F and 2′-OMe chemical modifications onboth strands arranged in proprietary positions and sequences canincrease knockdown by 50-fold and PS linkages at the ends greatlyincrease gene knockdown duration. We can design a small set of AsiCsbearing different covalent modifications of the siRNA portions of theAsiC and analyze their effect on knockdown of eGFP, AKT1 and PLK1

EpCAM-AsiCs targeting additional genes that we evaluate in vivo can bedesigned with the most active siRNA sequences and best chemicalmodifications. A small group of siRNA sequences to test for knockdown(without aptamers, by transfection) can be identified by web algorithms.The most efficient siRNAs (pM activity), which also have low predictedmelting temperatures (Tm), can be used, since these are processedbetter. If we need to use sequences with higher Tms, we can add amismatch at the 3′-end of the sense strand to promote siRNA unwindingand incorporation of the active strand in the RISC.

Eliminate off target effects and toxicity These experiments can beperformed with the original and the best optimized AsiCs. The lack oftoxicity of the various AsiCs encoding eGFP siRNA (whose knockdownshould not affect viability) can be formally assessed by Cell Titer-Gloassay of AsiC-incubated TNBC lines. Based on prior work, we do notexpect significantly reduced viability. Lipid transfection will be usedas a control for cytotoxic RNA delivery. Finally we can verify that eachof the AsiCs is not immunostimulatory by qRT-PCR, performed 6 and 24 hrpost AsiC incubation, to amplify a panel of inflammatory and innateimmune response genes (IFNB, IFNG, IL1, IL8, IL10, OAS1, STAT1, IP10).qRT-PCR is the most sensitive assay for immunostimulation and we chosetimes that capture the peak response. Cells treated with poly(I:C) canserve as positive controls and mock-treated cells will be negativecontrols. If any AsiC is immunostimulatory (a sequence and concentrationdependent property), we can evaluate whether additional chemicalmodifications, which reduce innate immune sensor binding, eliminateimmune stimulation without compromising gene knockdown. A 2′-F or 2′-OMemodification of the second residue of either the full AsiC or the Dicercleavage product can accomplish this goal.

Since the CD4-AsiC is not immunostimulatory in our prelim. studies andthe optimized AsiCs are active at greatly reduced concentrations (andoff-target effects are concentration dependent), innate immunestimulation is unlikely, but if detected, can be easily suppressed bychemical modification. In conjunction with the tissue explant studies wecan also examine tissue histology carefully for disruption of epithelialtissue architecture and cell necrosis.

Optimize tumor concentration and define PK/PD, Next we evaluate andimprove systemic T1/2 and tumor targeting in tumor-bearing mice. We canfocus on the original AsiC design and a few of the in vitro optimizedconstructs (as they are identified). We can use qRT-PCR to measurecirculating T1/2 and tissue distribution, in vivo imaging of thefluorescent AsiC to look at tumor localization and silencing of tumorcell mCherry (GFP is not used because of background autofluorescence) asa readout of gene silencing. Studies of EpCAM-AsiC PK/PD can befacilitated by our recent experience with in vivo imaging (FIGS. 16,17A-17B, and 18A-18B, data not shown). These experiments can use nudemice bearing mammary fatpad xenografts of Luciferase-mCherry stabletransfectants we have generated of EpCAM+ basal-A TNBC lines, such asMB468 or HCC1187, compared to an EpCAM− mesenchymal basal-B TNBC cellline, such as MB231. We have an expression plasmid for these tags anduse lentivirus infection to produce stable transfectants. ˜5-8 mice/gpwill be used to obtain statistical significance based on our prelim.data in these models. We can first compare the blood and tumorconcentration after iv and sc administration of the original AsiCconstruct and the constructs optimized for in vitro knockdown. Mice canbe examined frequently for clinical signs of toxicity. Samples can beanalyzed over 5d with frequent sample collection the first day. At eachtimepoint, blood and urine can be harvested and analyzed by Taqman assayfor the antisense strand. Tumor and sample organs can be harvested atfewer timepoints from euthanized animals. Blood can be analyzed forhematological, liver and kidney toxicity by blood counts and serumchemistries. The circulating T1/2 and proportion of the injected drugthat localizes to the EpCAM+ tumor can be calculated. Without wishing tobe bound by theory, based on our preliminary experiments with sc and ivadministration of the CD4-AsiCs and in vivo experience with thePSMA-AsiC, we expect that most of these EpCAM-AsiCs will be rapidlyexcreted after iv administration, but that sc injected EpCAM-AsiCs willconcentrate in tumor xenografts. The larger multimerized constructs(FIG. 20) might resist kidney filtration and have better tumorconcentration when given iv. The sc and iv PK results will be comparedwith mCherry knockdown following a single EpCAM-AsiC injection in arange of concentrations, assessed both by in vivo imaging (using theIVIS Spectrum) and by flow cytometry, FM, and qRT-PCR of tumor specimensharvested 4, 7 and 12 d post-treatment. These experiments can provideestimates of the effective dose required for peak tumor gene knockdownof 50, 75 and 90% (ED50, ED75, ED90) and for the durability of knockdownin the tumor (quantified as T-KD50=time for tumor expression to returnhalfway to control from the peak knockdown). These parameters can bedetermined for each chosen construct.

Next we assess ways to improve the circulating T1/2. These includeincreasing the size of the AsiC (i.e. by PEG conjugation comparing a fewsizes, such as 10, 20 and 30 kD, avoiding polymers known to be toxic,such as PEI) and increasing binding to serum proteins to reduce renalfiltration (i.e. by conjugation with cholesterol, which binds to serumLDL158,159 or by adding a diacyl tail to promote binding to serumalbumin. We avoid strategies that produce particles or aggregates sincethese will have poorer tumor penetration and may be trapped in theliver. Linking PEG to the 5′-end of the aptamer, the 3′-end of theinactive siRNA or the 3′-end of the active strand should not interferewith RNAi. In vivo PK/PD/toxicity evaluation can be performed as above,using the unconjugated AsiC as a positive control (and benchmark) andthe conjugated siRNA (without the aptamer) as a negative control. Two orthree of the constructs that have the lowest ED75 or ED90 and longestT-KD50 for GFP will be retested using a PLK1 EpCAM-AsiC to determine thecorresponding PK/PD parameters, to aid in designing the dosing regimenfor antitumor efficacy experiments. We can also determine the maximallytolerated dose (MTD) for these PLK1 constructs.

Antitumor Effect of EpCAM AsiCs against basal-like TNBCs Our final goalis to test the EpCAM-AsiCs against orthotopic mammary fat pad tumors andmetastases. We can use nude mice unless tumors do not grow or growslowly, in which case we will switch to NSG mice. Live animal imagingcan be performed using an IVIS Spectrum, sensitive for multicolorfluorescence and bioluminescence. These experiments can evaluate 2-3 ofthe best EpCAM-AsiCs identified.

Activity of PLK1 EpCAM-AsiCs against orthotopic xenografts We can beginby targeting PLK1/A few PLK1 EpCAM-AsiC designs, optimized as describedabove, can be injected sc and/or iv in groups of 5-8 mice (size chosenfrom power calculations based on previous experiments in which thisgroup size gave statistically significant results) using doses anddosing schedules/injection route chosen based on the PK/PD resultsabove. For example if the ED90 is well below the MTD, an initialexperiment might investigate administering 2ED90 every T-KD50/2 d. Micecan initially be treated as soon as their tumors become palpable, but inlater experiments we can investigate whether larger tumors of fixeddiameters regress after multiple administrations. Mice bearingrepresentative EpCAM+ basal-A (MB468, HC1187, BPLER) and EpCAM− basal-B(MB231) tumors will be compared. For some experiments, we can treat micebearing these tumors in each flank, but these may require more micebecause of intra-animal variations in tumor sizes. Control mice can betreated with PBS or naked siRNAs, the EpCAM aptamer on its own,EpCAM-AsiCs bearing scrambled siRNA sequences and PLK1 PSMA-AsiCs. Insome experiments we can compare EpCAM-AsiC treatment with adecatumumabor paclitaxel. Tumor size will be quantified by luminescence and calipermeasurements q3d. Treated mice can also be weighed and observed forclinical signs of toxicity and at time of sacrifice can be carefullyexamined for gut and bone marrow toxicity by blood counts andpathological examination of gut, bone marrow and spleen. Differencesbetween groups can be assessed by one way ANOVA with corrections formultiple comparisons as needed. For AsiCs that are effective, we canalso examine the immediate effect of treatment to evaluate the mechanismof antitumor activity and verify that the AsiCs are not activatinginnate immune responses. Tumor-bearing mice can be sacrificed 1-3 dafter a single therapeutic or control injection and the tumors stainedfor activated caspases to determine if death is by apoptosis and by H&Eto look for mitotic spindles to follow the expected effect of PLK1knockdown. Serum interferons and pro-inflammatory cytokines can beassessed by multiplexed ELISA, and spleen and tumor cells analyzed byqRT-PCR for the corresponding mRNAs. If there is no antitumor effect orthe antitumor effect is suboptimal, the dosing regimen can be adjustedto the MTD. If the antitumor effect is complete (complete tumorregression), then we can evaluate decreased doses and/or larger tumorsat start of therapy. When control mice are sacrificed because untreatedtumors have reached the allowed size, the treated mice can be sacrificedand mammary fatpads inspected for residual microscopic or macroscopictumor by FM, H&E and IHC. Residual tumor cells can also be assessed forEpCAM expression to determine whether tumor resistance, if it occurs,may have developed as a consequence of down-regulating EpCAM. If noresidual tumor cells are noted, we can perform an additional experimentto determine whether tumors are eradicated—mice will be treated for 1-2weeks after the luciferase measurement has returned to backgroundlevels, and then mice can be observed for 1-2 months off treatment tosee if tumors regrow or metastases appear. The most effective regimen(s)for basal-A TNBCs can also be evaluated against other breast cancersubtypes (luminal, Her2+) that we expect EpCAM to target.

PLK1 EpCAM-AsiC activity against metastatic tumors To evaluate theeffectiveness of EpCAM-AsiCs against metastatic cancer cells, we canevaluate the PLK1 EpCAM-AsiCs against basal-A TNBC cell lines injectedintravenously in NSG mice, which have the best tumor take. We can beginto treat mice as soon as lungs become luciferase+ after tail veininjection of basal-A (or basal-B as control) TNBCs. The treatment dosingcan use the effective schedule and mode of administration determinedabove for primary tumors. Mice can be imaged q3d. The controls can bereduced to a mock-treated group and groups treated with paclitaxel or anEpCAM-AsiC containing a non-targeting siRNA. When the control mice needto be sacrificed, all groups can be imaged. Lungs, livers and brains canbe dissected, weighed, imaged to quantify tumor burden, sections can beanalyzed by H&E and staining for EpCAM, and one lung from each animalwill be analyzed by qRT-PCR for relative expression of human/mouse Gapdhto quantify tumor burden independently. If mice treated with PLK1EpCAM-AsiCs are completely protected from metastases or show asignificant advantage compared to control groups, we can determine ifmice with greater metastatic burdens are also protected by delaying thebeginning of treatment until the tumor burden is greater.

We can also compare the most effective iv regimen with the mosteffective sc regimen identified above for treating orthotopic tumors,since RNA delivery/knockdown at metastatic sites could differ fromprimary tumor sites. We can also use this metastasis model to evaluatein vivo knockdown of our screen's BDG genes and genes identified aboveherein as necessary for tumor initiation ex vivo, since M-IC capabilityis thought to correlate with T-IC function.

Activity of EpCAM-AsiCs targeting BDF genes We can next compare PLK1knockdown with knockdown of TNBC dependency genes identified in oursiRNA screens or in the literature (such as XBP1). These in vivoexperiments for each gene target chosen can involve (1) identifyingactive siRNAs for each gene and evaluating the effect of knockdown oncell proliferation and T-IC function in vitro; (2) designing and invitro testing of AsiCs to knockdown the specific gene; (3) evaluatingthe effect of gene knockdown on in vitro proliferation and T-IC functionin a variety of breast cancer cell lines; and (4) verifying the lack ofoff-target immune stimulation of the individual AsiC. The genes thatbehave best in vitro can be advanced to in vivo testing in orthotopicand metastatic models as described above for PLK1. In these experimentswe can compare untreated mice with mice treated with EpCAM-AsiCstargeting the specific gene or PLK1. If there is a specific inhibitordrug for a particular gene target (i.e. bortezomib/carfilzomib for theproteasome), a group of control mice can also be treated with the drugfor comparison. Exemplary genes for such experiments are proteasomegenes and MCL1, U4/U6-U5 tri-snRNP complex genes96,97, XBP1 and thekinetochore gene NDC80. AsiCs that have the best in vivo activity ontheir own will also be evaluated in combinations with PLK1 AsiCs andeach other. Since proteasome inhibitor sensitivity correlates stronglywith MCL1 dependency in vitro (not shown), we hypothesize thatproteasome gene and MCL1 knockdown will be synergistic. The synergy ofdifferent AsiC and AsiC/drug combinations can be formally tested by theisobologram method using different RNA dose combinations or combinationswith relevant inhibitor drugs. In particular we will determine whethercombining EpCAM-AsiCs with standard of care drugs, such as paclitaxel,is synergistic with the original construct.

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Example 6

Material and Methods

Cell Culture

Human BPE and BPLER cells were grown in WIT medium (Stemgent). MB468were transduced with a luciferase reporter. All other human cell lineswere obtained from ATCC and grown in MEM (MCF7, BT474), McCoy's 5A(SKBR3), RPMI1640 (HCC1806, HCC1143, HCC1937, HCC1954, HCC1187, MB468,T47D) or DMEM (MB231, BT549, MB436) all supplemented with 10% FBS, 1 mML-glutamine and penicillin/streptomycin (Gibco) unless otherwiseindicated. 4T1 mouse breast cancer cells, were grown in 10% FBS DMEM.For in vivo imaging, MB468 cells stably expressing Firefly luciferase(MB468-luc) were used and MB231 cells stably expressing Fireflyluciferase and mCherry (MB231-luc-mCherry) were selected after infectionwith pLV-Fluc-mCherry-Puro lentivirus. MB231 Cells were selected withpuromycin.

For uptake and silencing treatment, cells were plated at low density(10,000 cells/well in 96-well plates) and treated immediately. All AsiCand siRNA treatments were performed in either OptiMEM or WIT medium.Cell viability was assessed by CellTiter-Glo (Promega) or by Trypan-Bluestaining in 96-well plates.

For colony formation assay, 1,000 viable cells were treated for 6 h inround bottom 96-well plates and then transferred to 10-cm plates inserum-containing medium. Medium was replaced every 3 d. After 8-14 d,cells were fixed in methanol (˜20C) and stained with crystal violet. Forsphere formation assay, 1,000/ml viable cells were treated for 6 h inround bottom 96-well plates and then cultured in suspension inserum-free DMEM/F121:1 (Invitrogen), supplemented with EGF (20 ng/ml, BDBiosciences), B27 (1:50, Invitrogen), 0.4% bovine serum albumin (Sigma)and 4 μg/ml insulin (Sigma). Spheres were counted after 1 or 2 weeks.

siRNA Transfection

Cells were transfected with Dharmafect I per the manufacturer'sprotocol. See below herein for all siRNA sequences.

Flow Cytometry.

For flow cytometry, cells were stained as previously described (Yu, F.et al (2007). let-7 Regulates Self Renewal and Tumorigenicity of BreastCancer Cells. Cell 131, 1109-1123. ), briefly, direct immunostaining ofEpCAM and AKT1 was performed using 1:50 dilutions of hAb for 30-60minutes at 4° C. (BioLegend/BD). Cells were stained in PBS containing0.5% FCS, 1 mM EDTA, and 25 mM HEPES. Samples were washed twice in thesame buffer. Data was acquired using FACS-Canto II (BD Biosciences).Analyses were performed in triplicate and 10,000 gated events/samplewere counted. All data analysis was performed using FlowJo (TreestarInc.).

RNA Analysis.

qRT-PCR analysis was performed as described (Petrocca, F., et al.(2008). E2F1-regulated microRNAs impair TGFbeta-dependent cell-cyclearrest and apoptosis in gastric cancer. Cancer Cell 13, 272-286).Briefly, total RNA was extracted with Trizol (Invitrogen) and cDNAprepared from 1000 ng total RNA using Thermoscript RT kit (Invitrogen)as per the manufacturer's SYBR Green Master Mix (Applied Biosystems) anda BioRad C1000 Thermal Cycler (Biorad). Relative CT values werenormalized to GAPDH and converted to a linear scale.

Collagenase Digestion of Human Breast Tissue.

Fresh breast or colon cancer and control biopsies were received from theUMASS Tissue Bank, samples were cut into 3×3×3 mm samples and placed ina 96well plate with 100 ul RPMI. Samples were treated with eitherAlexa647-siRNA-GFP, Alexa647-chol-siRNA-GFP or Cy3-AsiC-GFP for 24 hr.Samples were photographed and digested. Three samples from eachtreatment were pooled and put in 10 ml RPMI containing 1 mg/mlcollagenase II (Sigma-Aldrich) for 30 minutes at 37° C. with shaking.Samples were disrupted in a gentleMACS dissociator (Miltenyi) using thespleen program for 30 minutes at 37° C. both before and aftercollagenase digestion. Cell suspensions were passed through a 70-Gm cellstrainer (BD Falcon), washed with 30 ml RPMI, and stained for flowcytometry.

Animal Experiments

All animal procedures were performed with Harvard Medical School andBoston Children's Hospital Animal Care and Use Committee approval. Nudemice were purchased from the Jackson Laboratory.

In Vivo Experiments.

For tumor initiation studies 8-week old female Nu/J mice (Stock #002019,Jackson Laboratories) were injected subcutaneously with MB468-luc(5×10⁶) cells pretreated for 24 h with EpCAM-AsiC-GFP, EpCAM-AsiC-PLK1or untreated. Cells were trypsinized with Tryple Express (Invitrogen),resuspended in WIT media and injected subcutaneously in the flank.Following intraperitoneal injection of 150 mg/kg D-luciferin (CaliperLife Sciences) luminescent images of the whole body were taken every 5days for a total of 20 days using the IVIS Spectra system (Caliper LifeSciences).

For AsiC uptake experiments MB468-luc (5×10⁶) and MB231-luc-mCherry(5×10⁵) cells trypsinized with Tryple Express (Invitrogen), wereresuspended in a 1:1 WIT-Matrigel solution and injected subcutaneouslyin the flank of 8-week old female Nu/J mice (Stock #002019, JacksonLaboratories). Tumors size was analyzed daily using the IVIS Spectrasystem (Caliper Life Sciences). After 5 days tumors were clearly visibleand mice were injected subcutaneously in the neck area withAlexa750-EpCAM-AsiC-GFP (0.5 mg/kg). Localization of the AsiC comparedto the tumor was tested every 48 h for 7 days.

For tumor inhibition studies, MB468-luc (5×10⁶) and MB231-luc-mCherry(5×10⁵) cells trypsinized with Tryple Express (Invitrogen), resuspendedin a 1:1 WIT-Matrigel solution and injected subcutaneously in the flankof 8-week old female Nu/J mice (Stock #002019, Jackson Laboratories).Tumors size was analyzed daily using the IVIS Spectra, after 5 daystumors were clearly visible. Mice bearing tumors of comparable size wererandomized into 5 groups and treated with 5 mg/kg of EpCAM-AsiC-PLK1,EpCAM-AsiC-GFP, EpCAM-Aptamer, siRNA-PLK1 or untreated. Mice weretreated every 72 h for 14 days.

All Images were analyzed using Living Image® software (Caliper LifeSciences).

Statistical Analysis

Student's t-tests, computed using Microsoft Excel, were used to analyzethe significance between the treated samples and the controls where thetest type was set to one-tail distribution and two-sample equalvariance.

Results:

EpCAM-AsiC Specifically Targets Basal a Breast Cancer Cells

An EpCAM aptamer was selected by Systematic Evolution of Ligands byExponential Enrichment (SELEX) for binding to human EpCAM. The optimizedaptamer is only 19 nucleotides (nt) long and binds to human EpCAM with12 nM affinity (Shigdar S. et. al. RNA aptamer against a cancer stemcell marker epithelial cell adhesion molecule affinity Cancer Sci. 2011May; 102(5):991-8). It does not bind to mouse EpCAM (FIG. 22). Its shortlength is ideal for an AsiC drug, since RNAs of ˜60 nt or less in lengthcan be cheaply and efficiently chemically synthesized. The EpCAM-AsiCswe designed consist of a longer strand of 42-44 nt (19 nt aptamer+3 ntlinker+20-22 nt sense (inactive) strand of the siRNA), which is annealedto a 20-22 nt antisense (active) siRNA strand (FIG. 21A). Both strandswere commercially synthesized with 2′-fluoropyrimidine substitutions,which confer enhanced stability in serum and other bodily fluids(T1/2>>3 d) and prevent stimulation of innate immune RNA sensors. Wefirst assessed EpCAM cell surface levels by flow cytometry in a panel ofhuman breast cell lines (Table 2, FIG. 23). EpCAM was highly expressedby all basal A and luminal cancer cell lines tested, but not by basal Bcancer cell lines. EpCAM staining of normal human epithelial cells (BPE)was close to background, while its transformed derivative BPLER hadbright EpCAM staining (FIG. 21B). Several of a handful of designs tested(with the sense and antisense strands exchanged and several linkers)knocked down gene expression in EpCAM+, but not EpCAM−, cell lines, butthe design that worked best in dose response experiments is shown inFIG. 21A. To test whether EpCAM-AsiC will be specifically taken up byEpCAM+ cell lines we labeled the 3′ end of the antisense strand of theAsiC with Alexa647. BPLER basal A TNBC cell line overexpresses EpCAM,while BPE a control epithelial breast cell line do not (FIG. 21B). BothBPLER and BPE cell were treated with the Alexa647-EpCAM-AsiC targetingGFP, only BPLER displayed uptake of the AsiC (FIG. 21C). We furthervalidated the selective uptake of EpCAM-AsiC, by treating EpCAM+MDA-MB-468 cells and BPE controls with Cy3 labeled EpCAM-Aptamer (the 19nt aptamer was labeled with Cy3 at the 5′ end). After 22 and 43 hours weclearly saw selective AsiC uptake in EpCAM+ cells (data not shown). Tounderstand the ability of EpCAM AsiC to selectively trigger geneknockdown we chose BPLER and BPE cell lines which stably overexpressGFP. Cells were treated with either EpCAM-AsiCs targeting GFP ortransfected with GFP-siRNA as a positive control (FIG. 21D). Althoughtransfection with GFP-siRNAs knocked down gene expression equivalentlyin BPE and BPLER, EpCAM-AsiCs selectively knocked down expression inBPLER without any lipid; knockdown was uniform and comparable to thatachieved with lipid transfection.

These results clearly indicate that EpCAM-AsiC is selectively taken-upby EpCAM+ cell and can induce gene knockdown specifically in theseEpCAM+ cells. Also we show that using different fluorophores (Alexa647or Cy3) at different locations (5′ of aptamer or 3′ of anti-sensestrand) did not impact the specific uptake.

Specific mRNA and protein knockdown was further analyzed on 8 differentbreast cancer cells lines. Here we show that basal A and luminal celllines which overexpress EpCAM displayed decreased AKT1 mRNA and proteinlevels following treatment with EpCAM-AsiC targeting AKT1. Transfectionwith AKT1-siRNA had a similar knockdown effect on all cell lines, whileusing EpCAM-AsiC targeting GFP as a control did not effect any of thecell lines (FIG. 24A, 24B). There was a clear correlation between EpCAMexpression level and the knockdown effect both at an mRNA and proteinlevel (FIG. 24D, 24E).

To determine if human epithelial breast cancer tissue can specificallytake up EpCAM-AsiC compared to healthy human tissue. We tested humanepithelial breast cancer biopsies and healthy control tissue from thesame patient. Samples were treated for 24 h with Alexa647-siRNA-GFP,Alexa647-chol-siRNA-GFP or Cy3-EpCAM-AsiC-GFP (FIG. 25A). Human tumorsamples display higher EpCAM level as well as higher cytokeratin levels,an epithelial cell marker (FIG. 25B). Labeled siRNA and chol-siRNApenetrated both tumor and healthy tissue with similar efficacy whileEpCAM-AsiC was selectively uptaken by the tumor tissue and not by thehealthy control tissue sample (FIG. 25C, 25D). The uptake experiment wasrepeated in tumors from three different patients, each biopsy receivedwas tested 3 times for each treatment. A summary of all three patients(FIG. 25E). Colon cancer biopsies were tested and compared to matchedhealthy samples, both healthy and tumor colon samples were able to takeup Cy3-EpCAM-AsiC-GFP (FIG. 26)

EpCAM AsiC Targeting PLK1 Specifically Inhibits Cell Proliferation inBasal a Breast Cancer Cells

To understand whether EpCAM-AsiC can specifically target basal A andluminal breast cancer cells and inhibit proliferation we designed anEpCAM-AsiC targeting PLK1. PLK1 is a known trigger for G2/NI transition.The effect of EpCAM-AsiC targeting PLK1 on cell proliferation was testedon 10 breast cancer cells representative of basal A, B and luminal celllines. EpCAM-AsiC targeting PLK1 decreased cell proliferation in bothbasal A and luminal cell lines while having no effect on basal B cells(FIG. 27A). A correlation was seen between EpCAM expression levels andcell viability (FIG. 27B). To understand if EpCAM-AsiC will specificallytarget EpCAM+ cells in a mix cell population HCC1937 (EpCAM+GFP−) cellwere co-cultured with BPE (EpCAM-GFP+) cells and treated with EpCAM-AsiCtargeting PLK1 or untreated. Untreated co-culture displayed a similarration of cells (41% BPE and 59% HCC1937). Following EpCAM-AsiCtargeting PLK1 treatment the ratio of EpCAM+ cells decreased to 17% andEpCAM− cells increased to 83% indicating that the EpCAM-AsiCspecifically suppresses proliferation in EpCAM+ cells. The co-culturewas repeated with other basal A cell lines (MB468 and HCC1143) similarreults were obtained. When BPE cells were grown in a co-culture withbasal B cell (MB231) the ratio between BPE and MB231 cells stayed thesame regardless of the EpCAM-AsiC treatment (66% BPE and 33% MB231 inuntreated co-culture and 61% BPE and 38% MB231 following EpCAM-AsiCtreatment) (FIG. 27C, 27D).

To determine if the suppression effect of EpCAM-AsiC targeting PLK1 oncell viability in basal A cells is triggered by EpCAM-aptamer binding tothe EpCAM receptor or by silencing of PLK1 we treated cell with theEpCAM-aptamer and compared to EpCAM-AsiC targeting PLK1. EpCAM-AsiCtargeting PLK1 suppressed cell viability in basal A and luminal celllines while EpCAM-aptamer didn't effect cell viability in any of thecell lines (FIG. 28).

One of our goals was to understand if EpCAM-AsiC targeting PLK1 could beutilized to target T-ICs within a tumor. To examine whether it might beactive not only against the bulk of cells within basal-A and luminalcells, but also against the T-ICs within them, we treated basal A,B andluminal cell lines with EpCAM-AsiC targeting PLK1 for 24 hr and testedthe effect on in vitro colony and sphere formation. Basal A and luminalcell lines that form colonies when plated at clonal density (HCC1937,HCC1954, HCC1806 and MCF7) lost the ability to form colonies afterEpCAM-AsiC targeting PLK1 treatment, whereas resistant clones emergedafter paclitaxel treatment (FIG. FIG. 29A-29B). In contrast, exposure toEpCAM-AsiC targeting PLK1 did not effect colony formation of basal B(MB231 and BT549) cells, while paclitaxel had a similar effect to basalA and luminal cells, reducing colony formation but still resistantclones invariably emerged. Likewise, among breast cancer cell lines thatform spheres under non-adherent conditions, paclitaxel, reducedsphere-formation in all (FIG. 29C), while EpCAM-AsiC targeting PLK1specifically inhibited sphere formation in basal A and luminal. Toexamine whether pretreatment with EpCAM-AsiC targeting PLK1 will inhibitor delay tumor initiation in-vivo we treat MB-468-luc cell withEpCAM-AsiC targeting PLK1, GFP or untreated for 24 h and injected thecells into the flank of nude mice. Using the IVIS Spectra imaging systemwe followed tumor growth every 5 days for 20 days. Cells pretreated withEpCAM-AsiC targeting PLK1 did not show any sign of a tumor after 20 dayswhile untreated cells or cells pretreated with EpCAM-AsiC targeting GFPdisplayed tumors after 5 days and the tumor size grew during the 20 days(FIG. 29D).

EpCAM AsiC Targeting PLK1 Specifically Inhibits Tumor Initiation andGrowth in Basal a Breast Cancer Cells

We were able to show that EpCAM-AsiC can specifically target EpCAM+ cellin-vitro, to understand whether this ability is retained in-vivo wefirst tested the stability of EpCAM-AsiC in mouse and human serum overtime. We saw that EpCAM-AsiC is stable for at least 36 h in both mouseand human serum (FIG. 30A-30B). We injected nude mice with bothMB468-luc and MB231-luc-mCherry cells on opposite flanks. After 5 dayswhen tumors were clearly visible using the IVIS Spectra imaging system,we injected mice s.c. (in the neck area, as far away as possible fromthe tumor cells injection sight) with 0.5 mg/kg of Alexa750 labeledEpCAM-AsiC targeting GFP. The mice were imaged immediately afterinjection and again after 24, 48 hr and 5 days to follow the AsiClocalization. The Alexa750 labeled EpCAM-AsiC targeting GFP was clearlylocalized to the MB468-luc tumor (EpCAM+) and not the MB231-luc-mCherry(EpCAM−) tumor (FIG. 31A). Analysis of 7 mice indicates a significantincrease of Alexa750 in MB468 (EpCAM+) tumors (FIG. 31B). At day 5 thetumors were removed and visualized to validate that the Alexa750 labeledEpCAM-AsiC targeting GFP indeed entered the tumors. Increased level ofAlexa750 was negatively correlated with mCherry levels (data not shown)

Our cell viability and tumor initiation data indicates that EpCAM AsiCtargeting PLK1 specifically inhibits tumor growth in Basal A breastcancer cells. To test this hypothesis we injected nude mice with etherEpCAM− basal B cells (MB231-luc-mCherry cells) or EpCAM+ basal A cells(MB468-luc cells). Once tumors were clearly visible by the IVIS imagingsystem mice were treated with 5 mg/Kg of either EpCAM AsiC targetingPLK1 or GFP every 72 h for 14 days or left untreated. Mice were imagedusing the IVIS Spectra imaging system every 72 h for 14 days. MB468-luctumors treated with EpCAM-AsiC targeting PLK1 shrunk in size as early as6 days post treatment and in many mice completely disappeared after 14days, while MB231-luc-mCherry tumors remained unchanged. We believe thatEpCAM-AsiC did have some effect even though it was targeting GFP sincebasal A tumor treated with GFP AsiC did not increase in size as much ascontrol untreated mice. Treatment with EpCAM-Asic targeting GFP suppresstumor growth in both EpCAM+ and EpCAM-tumors but didn't eliminatetumors. Untreated tumors both EpCAM+ and EpCAM− increased in size overthe 14 days (FIG. 32A-32B).

TABLE 1 EpCAM-AsiC Sequences AsiC construct Sequence SEQ ID NOEpCAM PLK1 sense GCG ACU GGU UAC CCG GUC GUU  1UUG AAG AAG AUC ACC CUC CUU AdTdT EpCAM PLK1 anti-senseUAA GGA GGG UGA UCU UCU UCA  2 dTdT EpCAM AKT1 senseGCG ACU GGU UAC CCG GUC GUU 23 GCU GGA GAA CCU CAU GCU GdTdTEpCAM AKT1 anti-sense CAG CAU GAG GUU CUC CAG CdTdT 24 EpCAM GFP senseGCG ACU GGU UAC CCG GUC GUU 25 UGG CUA CGU CCA GGA GCG CAdTdTEpCAM GFP anti-sense UGC GCU CCU GGA CGU AGC CdTdT 26 siGFP senseUGG CUA CGU CCA GGA GCG 27 siGFP antisense UGC GCU CCU GGA CGU AGC 28siAKT1 sense GCU GGA GAA CCU CAU GCU G 29 siAKT1 antisenseCAG CAU GAG GUU CUC CAG C 30 siPLK1 sense UGA AGA AGA UCA CCC UCC UUA 31siPLK1 antisense UAA GGA GGG UGA UCU UCU UCA 32

TABLE 2 EpCAM mean fluorescence intensity (MFI) of human breast celllines EpCAM Cell line Subtype MFI BPE immortalized normal epithelium 2BPLER basal-A TNBC 109 HMLER unclassified TNBC (myoepithelial) 72HCC1143 basal-A TNBC 1068 HCC1937 basal-A TNBC 806 HCC1187 basal-A TNBC289 HCC1806 basal-A TNBC 558 HCC70 basal-A TNBC 443 MB468 basal-A TNBC340 MCF7 luminal 583 T47D luminal 799 BT549 basal-B TNBC 2 MB231 basal-BTNBC 31 MB436 basal-B TNBC 4 Human fibroblast Normal tissue 14

Example 7

Triple negative breast cancers have the worst prognosis of any breastcancer subtype and there is no targeted TNBC therapy. TNBCs have thephenotype associated with tumor initiating cells (T-IC), also known ascancer stem cells. T-IC are resistant to chemotherapy and thought to beresponsible for tumor relapse and metastasis.

EpCAM is expressed at gap junctions at low levels on normal epithelialcells, but much more highly expressed (100-1000-fold greater) throughoutthe membrane of virtually all epithelial cancers and is a known TI-Cmarker.

Described herein is a strategy for gene knockdown therapeutics forbasal-like TNBCs. As described herein, the aptamer-siRNA chimera (AsiC)platform is adapted to transfect epithelial breast cancer cells whilealso targeting breast tumor-initiating cells (T-IC). The aptamer bindsto EpCAM, highly expressed on cancer cells and cancer stem cells. Asproof-of-concept, the siRNA is directed at a kinase required for mitosisin all cells (PLK1).

As demonstrated herein, the EpCAM-AsiC's are stable in human and mouse.The EpCAM AsiCs can be chemically synthesized with 2′-F pyrimidines anddTdT at the 3′-ends, which makes them resistant to RNases and unlikelyto stimulate innate immunity.

Cells were treated with 4 mM EpCAM-AsiC for 5 days and specific AKT1protein silencing by AKT1-AsiC was detected by flow cytometry (FIG.24F).

MB468 tumors regress only after treatment with PLK1 EpCAM-AsiC. Micewith sc MB468 tumors were treated with 5 mg/kg RNA 2×/wk beginning whentumors became palpable. PLK1 EpCAM-AsiC, GFP SpCAM-AsiC, EpCAM aptamer,PLK1 siRNA, and mock treated samples were analyzed (FIG. 33)

What is claimed herein is:
 1. An aptamer-siRNA chimera (AsiC) chimericmolecule comprising an Epithelial Cell Adhesion Molecule (EpCAM) bindingaptamer domain and an inhibitory nucleic acid domain that inhibitsPolo-like Kinase 1 (Plk1) wherein the chimeric molecule comprises thesequence of SEQ ID NO: 1 or
 3. 2. The molecule of claim 1, wherein the3′ end of the molecule comprises dTdT.
 3. The molecule of claim 1,wherein the molecule comprises at least one 2′-F pyrimidine.
 4. Apharmaceutical composition comprising the molecule of claim 1 and apharmaceutically acceptable carrier.
 5. The composition of claim 4,comprising at least two chimeric molecules, wherein the chimericmolecules have different aptamer domains or inhibitory nucleic aciddomains.
 6. The composition of claim 5, wherein different apatmer orinhibitory nucleic acid domains recognize different targets.
 7. Thecomposition of claim 5, wherein different apatmer or inhibitory nucleicacid domains have different sequences and recognize the same target. 8.The composition of claim 4, further comprising an additional cancertreatment.
 9. The composition of claim 8, wherein the cancer treatmentis paclitaxel.